Compositions and methods for peptide expression and purification using a type iii secretion system

ABSTRACT

Disclosed are compositions and methods for expressing and purifying a peptide of interest using a Flagellar Type III secretion system. Disclosed are nucleic acid sequences that contain a FlgM nucleic acid sequence, a cleavage site, and a nucleic acid sequence of interest. Also disclosed are polypeptides that contain FlgM, a cleavage site and a peptide of interest. Methods of producing polypeptides that have FlgM, a cleavage site and a peptide of interest are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/404,919, 371(c) date Dec. 1, 2014, which is the National Stage ofInternational Application No. PCT/US2013/043384, filed May 30, 2013,which claims the priority benefit of U.S. Provisional Application No.61/689,284, filed May 30, 2012, each of which is hereby incorporated byreference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under GM062206 andGM48677 awarded by the National Institutes of Health. The government hascertain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Mar. 15, 2017, as a text file named“3694.0010002_SeqListing.txt,” created on Mar. 14, 2017, and having asize of 18,965 bytes, is hereby incorporated by reference pursuant to 37C.F.R. §1.52(e)(5).

FIELD OF THE INVENTION

The disclosed invention relates generally to the use of FlgM in a typeIII secretion system for expression of a peptide of interest.

BACKGROUND

Although large strides have been made in the recombinant expression ofproteins, the efficient expression of certain classes of proteinsremains a challenge. These include the small, highly stablepharmacologically active polypeptides with a high density of disulfidecrosslinks. A major group within this general class includes thepolypeptides present in animal venoms. Although several differentphylogenetic lineages have evolved venoms independently, allpolypeptides found have convergently evolved a common set of propertiesthat allow them to be exceptionally stable upon injection into anotherorganism. These polypeptides are of increasing interest because many ofthem have novel pharmacological activity and therefore serve as usefulligands in basic research or have direct diagnostic and therapeuticapplications. One of these peptides, MVIIA a 25 amino acid peptide withthree disulfide bonds, has become an approved drug for intractable pain.

When recombinant expression of small disulfide-rich polypeptides isattempted, the yields are generally low. A fundamental problem is thatwhen expression levels are high, the resulting high concentrations ofpolypeptide in the cell lead to the formation of intermolecularaggregates, and recombinant polypeptides are mostly found in inclusionbodies. The ability to recover the polypeptide from an inclusion body ina biologically active form is not predictable and requires additionalsteps that vary depending on the polypeptide expressed.

BRIEF SUMMARY

Disclosed herein are compositions and methods for overcoming the currentobstacles of production and purification of cysteine-rich polypeptides.The characterization of various factors of controlling flagellar geneexpression, ionic conditions, cell growth phase, and removal of cellularproteases or secretion competitors for the purpose of improving yield ofsecreted protein are disclosed.

Disclosed herein are methods of utilizing the flagellar FlgM protein asa vector for the secretion of small, highly stablepharmacologically-active polypeptides that contain a high density ofcysteine residues, which form disulfide crosslinks in the matureproduct. For example, a bacterial secretion system for the recombinantexpression of μ-conotoxin SIIIA in Salmonella typhimurium is provided.

Also disclosed herein are bacterial strains that can be used to producehigh yields of secreted protein for the purposes of protein purificationvia flagellar T3S.

Disclosed are compositions and methods for production and purificationof polypeptides using a bacterial flagellar system.

Disclosed are constructs comprising a FlgM nucleic acid sequence, acleavage site, and a nucleic acid sequence of interest. The constructscan further comprise a nucleic acid sequence encoding a purificationtag. The purification tag can be poly-histidine.

The FlgM nucleic acid sequence of the disclosed constructs can be wildtype FlgM. The cleavage site can be a Tobacco Etch Virus (TEV) proteasecleavage site or an Enterokinase (ETK) cleavage site.

The nucleic acid sequence of interest of the disclosed constructs canencode a cysteine-rich peptide. The cysteine-rich peptide can be aneuroactive toxin, such as a conopeptide. The conopeptide can be aJ-conotoxin such as SIIIA.

The disclosed constructs can have the cleavage site between the FlgMnucleic acid sequence and the nucleic acid sequence of interest. Theorder of the sequences in the constructs can be the FlgM nucleic acidsequence, the nucleic acid sequence encoding a purification tag, thecleavage site, and the nucleic acid sequence of interest. The order ofthe sequences in the constructs can be the nucleic acid sequenceencoding a purification tag, the FlgM nucleic acid sequence, thecleavage site, and the nucleic acid sequence of interest. The order ofthe sequences in the constructs can also include the nucleic acidsequence encoding a purification tag being C-terminal to the nucleicacid sequence of interest.

The disclosed constructs can also comprise a P_(ara)BAD promoter.

Also disclosed are polypeptides comprising FlgM, a cleavage site, and apeptide of interest. The polypeptides can further comprise apurification tag. The purification tag can be poly-histidine.

The FlgM in the disclosed polypeptides can be wild type FlgM. Thecleavage site can be a TEV protease cleavage site or an ETK cleavagesite.

The peptide of interest in the disclosed polypeptides can be acysteine-rich peptide. The cysteine-rich peptide can be a neuroactivetoxin such as a conopeptide. The conopeptide can be a μ-conotoxin suchas SIIIA.

The disclosed polypeptides can have the cleavage site between the FlgMand the peptide of interest. The order of the sequences in thepolypeptides can be the FlgM is N-terminal to the purification tag, thepurification tag is N-terminal to the cleavage site, and the cleavagesite is N-terminal to the peptide of interest. The order of thesequences in the polypeptide can be the purification tag is N-terminalto FlgM, FlgM is N-terminal to the cleavage site, and the cleavage siteis N-terminal to the peptide of interest. In some aspects, thepurification tag is C-terminal to the peptide of interest.

Also disclosed are recombinant cell lines comprising any of thedisclosed constructs. The recombinant cell line can be derived from awild type strain of Salmonella enterica serovar Typhimurium.

The genome of the disclosed recombinant cell lines can comprise analteration to one or more flagellin or hook-associated protein genes.The one or more flagellin genes can be selected from the groupconsisting of flgK, flgL, fliC, fljB, and fliD.

The disclosed recombinant cell lines can comprise an alteration to oneor more inhibitors of the flagellar FlhD4C2 master regulatory proteincomplex. The inhibitors of the flagellar FlhD4C2 master regulatoryprotein complex can be selected from the group consisting of fimZ, srgD,hdfR, rbsR, ompR, clpX clpP, lrhA, ydiV, dskA, ecnR, fliT, and rcsB.

The disclosed recombinant cell lines can comprise a mutation to increasetranscription or translation of the FlgM T3S-chaperone gene fliA.

Also disclosed are methods of producing a peptide of interest comprisingculturing a cell line comprising any of the disclosed polypeptides inculture media, wherein the polypeptide comprises the peptide ofinterest. The methods can further include purifying the peptide ofinterest from the culture media. The methods can use a cell line thatcomprises any of the disclosed constructs.

The purifying of the peptide of interest can comprise an affinity columnsuch as σ²⁸ affinity column.

The disclosed methods can use a cell line that comprises a flagellartype III secretion (T3 S) system of Salmonella enterica serovarTyphimurium to secrete the polypeptide comprising the peptide ofinterest.

The disclosed methods can use a cell line that comprises an alterationto one or more flagellin hook-associated protein genes. The one or moreflagellin or hook-associated protein genes can be selected from thegroup consisting of flgK, flgL, fliC, fljB, and fliD.

The disclosed methods can use a cell line that comprises an alterationto one or more inhibitors of the flagellar FlhD4C2 master regulatoryprotein complex. The inhibitors of the flagellar FlhD4C2 masterregulatory protein complex can be selected from the group consisting offimZ, srgD, hdfR, rbsR, ompR, clpX, clpP, lrhA, ydiV dskA, ecnR, fliT,and rcsB.

The disclosed methods can use a cell line that comprises a mutation toincrease transcription or translation of the FlgM T3S-chaperone genefliA.

Additional advantages of the disclosed method and compositions will beset forth in part in the description which follows, and in part will beunderstood from the description, or may be learned by practice of thedisclosed method and compositions. The advantages of the disclosedmethod and compositions will be realized and attained by means of theelements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of thedisclosed method and compositions and together with the description,serve to explain the principles of the disclosed method andcompositions.

FIGS. 1 A-C show engineering of a flagellar type III secretion systemfor the secretion of SIIIA conotoxins. FIG. 1A. Model: A FlgM-SIIIAtranslational fusion is a secretion substrate of the bacterial T3SS. Thefusion construct is secreted via the flagellar-specific T3SS through theflagellar channel into the culture medium through flagellar structuresthat are competent for FlgM secretion. Expression of FlgM-SIIIA isinduced upon addition of arabinose and is independent of flagellar classI and II gene expression. During HBB assembly, FlgM remains inside thecytosol and acts as an anti σ28-factor preventing transcription of classIII genes, e.g. genes encoding for the flagellin subunit FliC or thestator proteins MotAB. The HBB structure is completed within 30 min,which coincides with a substrate specificity switch within the flagellarsecretion apparatus (indicated by an orange asterisk in the figure) fromearly to late-substrate secretion. This results in secretion of FlgM andsubstrates needed during the final phase of flagella assembly. OM: outermembrane, PG: peptidoglycan layer, IM: inner membrane. FIG. 1B.Expression and secretion of FlgM-SIIIA fusion protein. SecretedFlgM-SIIIA was precipitated using TCA and immunoblots with antibodiesagainst FlgM are shown for cellular and supernatant fractions. Proteinbands of native FlgM (open triangle) and FlgM-SIIIA (filled triangle)fusions are marked next to the blot. Construct 1-3 (labeled c1-c3)represent the following protein fusions: c1=H6-FlgM-TEV-SIIIA,c2=FlgM-TEV-SIIIA-H6, c3=FlgM-H6-TEV-SIIIA. Secretion efficiencies ofthree FlgM-SIIIA constructs varying in their position of thepoly-histidine tag were tested. Secretion levels are shown for TH437(wt, lane 1), TH4885 (ΔfliF, lane 2), TH5139 (ΔFlgM, lane 3), TH10874(Para::FlgM-FKF, lane 4), TH15705 (fliA* Para::construct 1, lane 5),TH15706 (fliA* Para::construct 2, lane 6), and TH15707 (fliA*Para::construct 3, lane 7). Wildtype FlgM bands in lane 5-7 were visibleupon extended exposure. FIG. 1C. FlgM-H6-TEV-SIIIA was expressed fromthe arabinose promoter and secretion was compared in a FlgM (lane 2),fliA* (H14D, lane 3) and ΔfliCD (lane 4) background. Secretion ofwildtype FlgM expressed from its native promoter is shown in lane 1(TH437, labeled wt).

FIGS. 2 A-D show purification and electrophysiology of recombinantconotoxin SIIIA and secretion of toxins from various organisms via theflagellar T3SS. FIG. 2A. The supernatant of a strain expressing andsecreting recombinant SIIIA fused to FlgM (TH15707 fliA* (H14D)ΔaraBAD1036::FlgM-H6-TEV-SIIIA) was filtered and bound to a Ni2+-IDAcolumn as described in Example 1. The matrix was washed after binding(W1-W3) and FlgM-SIIIA was eluted in three steps withimidazole-containing elution buffer (E1-E3). For Western blot detection,samples were TCA precipitated. Due to the increased FlgM-SIIIAconcentration in the elution fractions, only 1/10th of the volume wasused for TCA precipitation of elution fractions 1-3. FIG. 2B.Recombinant SIIIA blocks voltage-gated sodium channel NaV1.2. A Xenopusoocyte expressing rat NaV1.2 was exposed to 10 μM rSIIIA while sodiumcurrents were monitored as described in Example 1. Currents recordedbefore toxin exposure (control, gray trace), and following 20-minexposure to 10 μM rSIIIA (black trace). Each trace represents theaverage of five responses. The difference of the peak values between thetwo traces corresponds to the inhibitory effect rSIIIA has on channel NaV1.2. FIG. 2C. FlgM-H6-TEV was translationally fused to six differenttoxins from cone snails, and one toxin each from sea anemone, scorpion,spider, and snake, and FlgM-H6 was fused to the Corynebacteriumdiphtheria (see also Table 3 for detailed list). FIG. 2D. Toxins wereexpressed in a Salmonella poly-hook background. Secretion of diphtheriatoxin fragment A from Salmonella strain TH16229 was tested using threeindependent biological replicates (labeled 1-3). Secretion ofrecombinant toxins was performed as described before.

FIGS. 3 A-B show the effect of Salmonella poly-hook background onsecretion. FIG. 3A. Immunostaining of the poly-hook background TH16778used for secretion of FlgM-H6-TEV-toxin fusions. Top left: Membranestained with FM-64. Top right: DNA stained with Hoechst. Bottom left:Flagellar hook-basal-body complexes (flgE::3×HA) labeled withanti-hemagglutinin antibodies coupled to Alexa Fluor-488. Scale bar is 5μm. FIG. 3B. Secreted proteins were precipitated using TCA andimmunoblots with antibodies against FlgM and FliK are shown for cellularand supernatant fractions. Protein bands of native FlgM (open triangle)and FlgM-SIIIA fusions (filled triangle) are marked next to the blot.Secretion levels were compared by measuring the densitometric intensityof the detected bands using ImageJ. Secretion values relative to thesecretion in a Para wildtype background (first lane) are displayed belowthe corresponding band.

FIG. 4 shows FlgM secretion under ParaBAD over-expression conditions.Western blot analysis of supernatant fractions of strainsover-expressing FlgM from the chromosomal araBAD promoter(ParaBAD-FlgM+). Overnight cultures were diluted 100-fold into LBmedium, incubated at 37° C. for 2 hours, followed by addition ofarabinose to 0.2% to induce ParaBAD-FlgM+. After 5 hours furtherincubation at 37° C., cells were separated from the supernatant bycentrifugation and TCA was added to the cell supernatant to precipitatesecreted protein (see Example 2). For the secreted protein analysis, 100OD units of sample were loaded into each lane. For whole cell proteinanalysis, 50 OD units of sample were loaded. Anti-FlgM antibody was usedto determine levels of secreted and whole cell FlgM (secreted FlgM=SeFlgM, Whole cell FlgM=WC FlgM, 0.2% arabinose=Ara).

FIGS. 5 A-D show the effect of flhDC operon expression on levels ofsecreted FlgM and cell motility. FIG. 5A. Secreted levels of FlgM wasdetermined in strains affected in flhDC operon expression byquantitative Western blot analysis using anti-FlgM antibody to detectedFlgM in the supernatant of the spent growth medium. (secreted FlgM=SeFlgM, cellular soluble FlgM=So FlgM, cellular insoluble FlgM=Inso FlgM,and whole cellular DnaK=WC DnaK). FIG. 5B. Relative FlgM secretedlevels. FIG. 5C. Swim phenotype on soft agar plates of strain TH18649(ΔPflhDC8089::tetR-PtetA ΔaraBAD1156::FlgM+) with no inducers added, 1μg/ml anyhdrotetracycline (ATc,) added and both ATc and 0.2% arabinose(Ara) added. FIG. 5D. Motility assay in various mutant backgroundsdepicted with ΔaraBAD1156::FlgM+ in the absence and presence of added0.2% arabinose.

FIGS. 6 A-C show the effect of fliA(σ28) alleles on levels of secretedFlgM and cell motility. FIG. 6A. Secreted levels of FlgM was determinedin strains carrying different alleles of the σ⁸ structural gene fliA byquantitative Western blot analysis using anti-FlgM antibody to detectedFlgM in the supernatant of the spent growth medium. (secreted FlgM=SeFlgM, whole cellular FlgM=WC FlgM, whole cellular FliA=WC FliA, andwhole cellular DnaK=WC DnaK). FIG. 6B. Relative FlgM secreted levels.FIG. 6C. Motility assay in various mutant backgrounds depicted withΔaraBAD1156::FlgM+ in the absence and presence of added 0.2% arabinose.

FIGS. 7 A-B show the effect of flagellar late substrate deletion, growthphase, and fljBenx vh2 mutations on levels of secreted FlgM and cellmotility. FIG. 7A. Secreted levels of FlgM was determined in strainscarrying different flagella late substrate genes (flgK, flgL, fliC, fliDand fljB) deletion mutants, growth phase (Δhin-5717, Δhin-5718) mutantsand fljBenx vh2 mutants by quantitative Western blot analysis usinganti-FlgM antibody to detected FlgM in the supernatant of the spentgrowth medium. (secreted FlgM=Se FlgM, cellular soluble FlgM=So FlgM,cellular insoluble FlgM=Inso FlgM, and whole cellular DnaK=WC DnaK).FIG. 7B. Relative FlgM secreted levels.

FIGS. 8 A-B show the effects of late flagella T3 S chaperone deletionmutations on levels of secreted FlgM and cell motility. FIG. 8A.Secreted levels of FlgM was determined in strains carrying differentdeletion mutant alleles of the flagellar T3 S chaperone genes flgN, fliSand fliT structural genes by quantitative Western blot analysis usinganti-FlgM antibody to detected FlgM in the supernatant of the spentgrowth medium. (secreted FlgM=Se FlgM, whole cellular FlgM=WC FlgM, andwhole cellular DnaK=WC DnaK). FIG. 8B. Relative FlgM secreted levels.

FIGS. 9 A-B show the effects of Spi-1 and Spi-2 mutations deletionmutations on levels of secreted FlgM and cell motility. FIG. 9A.Secreted levels of FlgM was determined in strains carrying deletionmutant alleles of the either the Spi1 or Spi2 genes by quantitativeWestern blot analysis using anti-FlgM antibody to detected FlgM in thesupernatant of the spent growth medium. (secreted FlgM=Se FlgM, wholecellular FlgM=WC FlgM, and whole cellular DnaK=WC DnaK). FIG. 9B.Relative FlgM secreted levels.

FIGS. 10 A-C show the effects of cellular protease mutant alleles onlevels of secreted FlgM and cell motility. FIG. 10A. Secreted levels ofFlgM was determined in strains carrying deletion mutant alleles of theompT, degP, clpA, clpX or clpP genes with and without a functional flhDCoperon by quantitative Western blot analysis using anti-FlgM antibody todetected FlgM in the supernatant of the spent growth medium. (secretedFlgM=Se FlgM, whole cellular FlgM=WC FlgM, and whole cellular DnaK=WCDnaK). FIG. 10B. Relative FlgM secreted levels in the flhD+C+ and flhDCnull backgrounds, and relative whole cell FlgM accumulation level inflhDC null background. FIG. 10C. Motility assay in various mutantbackgrounds depicted with ΔaraBAD1156:FlgM+ in the absence and presenceof added 0.2% arabinose.

FIGS. 11 A-C show the effects of NaCl and KCl ionic strengths on levelsof secreted FlgM and cell motility. FIG. 11A. Secreted levels of FlgMwas determined in medium with NaCl and KCl added at concentrationsdepicted by quantitative Western blot analysis using anti-FlgM antibodyto detected FlgM in the supernatant of the spent growth medium.(secreted FlgM=Se FlgM, cellular soluble FlgM=So FlgM, cellularinsoluble FlgM=Inso FlgM, and whole cellular DnaK=WC DnaK). FIG. 11B.Relative FlgM secreted levels. FIG. 11C. Motility assay in variousmutant backgrounds depicted with ΔaraBAD1156::FlgM+ in the absence andpresence of added 0.2% arabinose.

FIGS. 12 A-B show the effects of different combined mutations on levelsof secreted FlgM. FIG. 12A. Secreted levels of FlgM was determined instrains carrying different combined mutations depicted by quantitativewestern blot analysis using anti-FlgM antibody to detected FlgM in thesupernatant of the spent growth medium (secreted FlgM=Se FlgM, wholecellular FlgM=WC FlgM, and whole cellular DnaK=WC DnaK). FIG. 12B.Relative FlgM secreted levels.

FIGS. 13 A-B show the effects of different combined mutations on levelsof secreted FlgM-6H-TEV-δ-SVIE and FlgM-6H-ETK-δ-SVIE. FIG. 13A.Secreted levels of FlgM-6His-TEV-δ-SVIE and FlgM-6His-ETK-δ-SVIE wasdetermined in strains carrying different combined mutations depicted byquantitative western blot analysis using anti-FlgM antibody to detectedFlgM-6His-TEV-δ-SVIE and FlgM-6His-ETK-δ-SVIE in the supernatant of thespent growth medium (secreted FlgM-6His-TEV-δ-SVIE=SeFlgM-6His-TEV-SVIE, secreted FlgM-6His-ETK-δ-SVIE=SeFlgM-6His-ETK-δ-SVIE, secreted FlgM=Se FlgM, and whole cellular DnaK=WCDnaK). FIG. 13B. Relative FlgM-6His-TEV-δ-SVIE and FlgM-6His-ETK-δ-SVIEsecreted levels.

FIG. 14 shows type III secreted proteins. A coomassie-stained SDS gelfrom the spent growth medium of Salmonella cells in wild-type and mutantstrains was run. Lane 1 wild-type, lane 2 mot, lane 3 fliK, lane 4 flhD,lane 5 flhD invA.

FIGS. 15 A-E show the Salmonella SPI1 injectisome system. FIG. 15A. Allgenes required for the structure and assembly of the SPI1 injectisomeare clustered in the chromosome. FIG. 15B. SPI1 genes are under controlof the flagellar master operon flhDC, which leads to the successivetranscription of the FliZ, HilD and HilA regulators and HilA is requiredfor injectisome gene transcription. FIG. 15C. Injectisome basal body(IBB) completion is followed by FIG. 15D, needle and FIG. 15E,translocon assembly.

FIG. 16 shows flagellar and SPI1 injectisome structures. The structuresshow overall similarities in structure with strong homologies to basaland secretion-associated component proteins.

DETAILED DESCRIPTION

The disclosed method and compositions may be understood more readily byreference to the following detailed description of particularembodiments and the Example included therein and to the Figures andtheir previous and following description.

It is to be understood that the disclosed method and compositions arenot limited to specific synthetic methods, specific analyticaltechniques, or to particular reagents unless otherwise specified, and,as such, may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

A. Definitions

It is understood that the disclosed method and compositions are notlimited to the particular methodology, protocols, and reagents describedas these may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present invention which willbe limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present method andcompositions, the particularly useful methods, devices, and materialsare as described. Publications cited herein and the material for whichthey are cited are hereby specifically incorporated by reference.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such disclosure by virtue of priorinvention. No admission is made that any reference constitutes priorart. The discussion of references states what their authors assert, andapplicants reserve the right to challenge the accuracy and pertinency ofthe cited documents. It will be clearly understood that, although anumber of publications are referred to herein, such reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “apolypeptide” includes a plurality of such polypeptides, reference to“the cell line” is a reference to one or more cell lines and equivalentsthereof known to those skilled in the art, and so forth.

“Optional” or “optionally” means that the subsequently described event,circumstance, or material may or may not occur or be present, and thatthe description includes instances where the event, circumstance, ormaterial occurs or is present and instances where it does not occur oris not present.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, also specifically contemplated and considered disclosed isthe range from the one particular value and/or to the other particularvalue unless the context specifically indicates otherwise. Similarly,when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another,specifically contemplated embodiment that should be considered disclosedunless the context specifically indicates otherwise. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint unless the context specifically indicates otherwise. Finally,it should be understood that all of the individual values and sub-rangesof values contained within an explicitly disclosed range are alsospecifically contemplated and should be considered disclosed unless thecontext specifically indicates otherwise. The foregoing appliesregardless of whether in particular cases some or all of theseembodiments are explicitly disclosed.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.In particular, in methods stated as comprising one or more steps oroperations it is specifically contemplated that each step comprises whatis listed (unless that step includes a limiting term such as “consistingof”), meaning that each step is not intended to exclude, for example,other additives, components, integers or steps that are not listed inthe step.

The term “vector” refers to a nucleic acid sequence capable oftransporting into a cell another nucleic acid to which the vectorsequence has been linked. The term “expression vector” includes anyvector, (e.g., a plasmid, cosmid or phage chromosome) containing a geneconstruct in a form suitable for expression by a cell (e.g., linked to atranscriptional control element). “Plasmid” and “vector” are usedinterchangeably, as a plasmid is a commonly used form of vector.Moreover, the invention is intended to include other vectors which serveequivalent functions.

The term “sequence of interest” or “nucleic acid sequence of interest”can mean a nucleic acid sequence (e.g., gene capable of encoding acysteine-rich peptide), that is partly or entirely heterologous, i.e.,foreign, to a cell into which it is introduced.

The term “sequence of interest” or “nucleic acid sequence of interest”can also mean a nucleic acid sequence, that is partly or entirelyhomologous to an endogenous gene of the cell into which it isintroduced, but which is designed to be inserted into the genome of thecell in such a way as to alter the genome (e.g., it is inserted at alocation which differs from that of the natural gene or its insertionresults in “a knock-in”). For example, a sequence of interest can becDNA, DNA, or mRNA.

A “peptide of interest” or “protein of interest” means a peptide orpolypeptide sequence (e.g., a cysteine-rich peptide), that is expressedfrom a sequence of interest or nucleic acid sequence of interest.

The term “operatively linked to” refers to the functional relationshipof a nucleic acid with another nucleic acid sequence. Promoters,enhancers, transcriptional and translational stop sites, and othersignal sequences are examples of nucleic acid sequences operativelylinked to other sequences. For example, operative linkage of DNA to atranscriptional control element can refer to the physical and functionalrelationship between the DNA and promoter such that the transcription ofsuch DNA is initiated from the promoter by an RNA polymerase thatspecifically recognizes, binds to and transcribes the DNA.

The terms “transformation” and “transfection” mean the introduction of anucleic acid, e.g., an expression vector, into a recipient cellincluding introduction of a nucleic acid to the chromosomal DNA of saidcell.

Also disclosed are transcriptional control elements (TCEs). TCEs areelements capable of driving expression of nucleic acid sequencesoperably linked to them. The constructs disclosed herein comprise atleast one TCE. TCEs can optionally be constitutive or regulatable.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed method and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a construct is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the construct are discussed, each and every combination andpermutation of the construct and the modifications that are possible arespecifically contemplated unless specifically indicated to the contrary.Thus, if a class of molecules A, B, and C are disclosed as well as aclass of molecules D, E, and F and an example of a combination molecule,A-D is disclosed, then even if each is not individually recited, each isindividually and collectively contemplated. Thus, is this example, eachof the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F arespecifically contemplated and should be considered disclosed fromdisclosure of A, B, and C; D, E, and F; and the example combination A-D.Likewise, any subset or combination of these is also specificallycontemplated and disclosed. Thus, for example, the sub-group of A-E,B-F, and C-E are specifically contemplated and should be considereddisclosed from disclosure of A, B, and C; D, E, and F; and the examplecombination A-D. This concept applies to all aspects of this applicationincluding, but not limited to, steps in methods of making and using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed it is understood that each of these additionalsteps can be performed with any specific embodiment or combination ofembodiments of the disclosed methods, and that each such combination isspecifically contemplated and should be considered disclosed.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the following claims.

B. Bacteria Flagella

Many bacteria utilize flagella to move in a directed manner, either awayfrom stressful environments or towards nutrients, O₂, light and otherpositive stimuli. The bacterial flagellum is a complex cellular machinethat requires more than 30 gene products for its construction. ForSalmonella enterica there are currently more than 60 genes involved inthe biogenesis and function of its flagella. These genes are organizedinto a transcriptional hierarchy of 3 promoter classes. At the top ofthe flagellar transcriptional hierarchy is the flhDC operon encoding themaster regulator proteins FlhD and FlhC, which form a heteromultimeric,transcriptional activation complex. The FlhD4C2 complex directs σ⁷⁰ RNApolymerase to transcribe from class 2 flagellar promoters. Class 2flagellar genes encode proteins required for the structure and assemblyof a rotary motor called the hook-basal body (HBB), a key structuralintermediate in flagellum assembly. The HBB includes the flagellar typeIII secretion (T3 S) system, which exports flagellar proteins from thecytoplasm through the growing structure during assembly. In addition toHBB gene expression, flagellar class 2 transcription produces σ²⁸ (FliA)and FlgM. These are regulatory proteins that couple transcription of theflagellar class 3 promoters to completion of the HBB. The σ²⁸ protein isa flagellar-specific transcription factor that directs RNA polymerase totranscribe from the flagellar class 3 promoters. Class 3 genes includethe structural genes of the flagellar filament and genes of thechemosensory signal transduction system that controls the direction offlagellar rotation according to changing concentrations of extracellularligands. Prior to HBB completion, FlgM binds σ²⁸ and prevents flagellarclass 3 promoter transcription. Upon HBB completion, a change in theflagellar T3S substrate specificity results in FlgM secretion andinitiation of σ²⁸-dependent transcription from flagellar class 3promoters. The secretion signal requirements for T3S substrates remainspoorly defined, but all substrates utilize an N-terminal peptidesecretion signal that is disordered in structure and unlike type IIsecretion, is not cleaved during the secretion process. Substratesecretion is often facilitated by T3S chaperone-assisted delivery to thesecretion apparatus. The FlgM protein is 97 amino acids in length andits secretion is dependent on an N-terminal secretion signal. FlgMsecretion is greatly enhanced by its secretion chaperone, σ²⁸, whichbinds to the C-terminal half of FlgM.

Because FlgM is a small T3S substrate and not part of the finalflagellar structure, it can be used as a vehicle to direct secretion ofproteins for purification purposes. Fusion of foreign peptides to theC-terminus of FlgM can be used to direct their secretion either into theperiplasm or into the extracellular milieu. The FlgM type Ill secretionsystem can be used to express and purify recombined proteins.

Disclosed are constructs and methods that use an expression system thatexploits the flagellar secretion system of Salmonella enterica serovarTyphimurium (Salmonella typhimurium) and bypasses the inclusion bodyproblem of recombinant small peptide expression.

C. Nucleic Acids Constructs

Disclosed are nucleic acid constructs comprising a FlgM nucleic acidsequence, a cleavage site, and a nucleic acid sequence of interest. Theconstructs can further comprise a nucleic acid sequence encoding apurification tag.

The order of the FlgM nucleic acid sequence, the cleavage site, and thenucleic acid sequence of interest can vary. In some aspects, the orderof the sequences can be from 5′ to 3′, the FlgM nucleic acid sequence,the nucleic acid sequence encoding a purification tag, the cleavagesite, and the nucleic acid sequence of interest. In some aspects, theorder of the sequences can be from 5′ to 3′, the nucleic acid sequenceencoding a purification tag, the FlgM nucleic acid sequence, thecleavage site, and the nucleic acid sequence of interest. In someaspects, the order of the sequences can be from 5′ to 3′, the FlgMnucleic acid sequence, the cleavage site, the nucleic acid sequenceencoding a purification tag, and the nucleic acid sequence of interest.In some aspects, the order of the sequences can be from 5′ to 3′, theFlgM nucleic acid sequence, the cleavage site, the nucleic acid sequenceof interest, and the nucleic acid sequence encoding a purification tag.Thus, the nucleic acid sequence encoding a purification tag can be 5′ or3′ to the nucleic acid sequence of interest.

1. FlgM

The disclosed constructs comprise a FlgM nucleic acid sequence. The FlgMnucleic acid sequence can be wild type FlgM. In some aspects, the FlgMnucleic acid sequence can be a mutant sequence of FlgM. The mutantsequence of FlgM can have one or more nucleotide mutations compared towild type FlgM. In some aspects, the mutations do not change the encodedamino acid sequence. In some aspects, the mutations in the mutantnucleic acid sequence of FlgM does not affect the ability of the encodedFlgM peptide to act as a vector for the secretion of the peptide encodedby the nucleic acid sequence of interest.

2. Cleavage Site

The disclosed constructs can comprise a cleavage site between the FlgMnucleic acid sequence and the nucleic acid sequence of interest. Thecleavage site can be a Tobacco Etch Virus (TEV) protease cleavage siteor an Enterokinase (ETK) cleavage site. Other cleavage sites known tothose of skill in the art can be used. Although the cleavage site isbetween the FlgM nucleic acid sequence and the nucleic acid sequence ofinterest, the cleavage site does not always have to be contiguous withthose sequences. In other words, a sequence encoding a purification tagcan be directly before or after the cleavage site.

The cleavage site can be a protease cleavage site. Therefore, thenucleic acid sequence of the cleavage site can encode a proteasecleavage site. The cleavage site is not a nuclease cleavage site andthus the nucleic acid sequences present in the constructs are notcleaved. The cleavage site allows for cleavage of the polypeptideencoded by the disclosed constructs. Cleavage of the polypeptide encodedby the disclosed constructs can release the peptide of interest (encodedby the nucleic acid of interest) from FlgM peptide (encoded by the FlgMnucleic acid sequence).

3. Nucleic Acid Sequence of Interest

The disclosed constructs comprise a nucleic acid sequence of interest.The nucleic acid sequence of interest can encode a peptide of interestto be expressed and purified using the FlgM system provided herein.

In some aspects, the nucleic acid sequence of interest encodes acysteine-rich peptide or a disulfide-rich peptide. Recombinantexpression of small disulfide-rich polypeptides results in generally lowyields. Overexpression of these polypeptides can lead to the formationof intermolecular aggregates, and the recombinant polypeptides can befound in the inclusion bodies. Because recovering the polypeptides fromthe inclusion bodies can be difficult and time consuming, thesedisulfide-rich polypeptides are best purified using the FlgM expressionsystem disclosed herein.

In some aspects, the nucleic acid sequence of interest encodes acysteine-rich peptide or a disulfide-rich peptide, wherein thedisulfide-rich or cysteine-rich polypeptides is a neuroactive toxin. Theneuroactive toxin can be any neuroactive toxin. In some aspects, theneuroactive toxin can be a conoidean derived toxin (i.e. a toxin from aconoidean). In some aspects, the neuroactive toxin can be a conopeptide.The conopeptide can be a μ-conotoxin. Examples of μ-conotoxins includebut are not limited to SIIIA.

4. Purification Tag

The disclosed constructs can further comprise a sequence encoding apurification tag. Examples of purification tags include, but are notlimited to poly-histidine, glutathione S-transferase (GST), Myc, HA,FLAG, and maltose binding protein (MBP). Thus, nucleic acid sequencesthat encode these purification tags are disclosed. Purification tags andthe nucleic acid sequences that encode them are well known in the art.

5. Promoter

The disclosed constructs can also comprise transcriptional controlelements (TCEs). TCEs are elements capable of driving expression ofnucleic acid sequences operably linked to them. The constructs disclosedherein comprise at least one TCE. TCEs can optionally be constitutive orregulatable. For example, disclosed herein are constructs comprising aP_(araBAD) promoter. The P_(araBAD) promoter is an arabinose-induciblepromoter. This allows for expression of the nucleic acid sequencesdownstream of the promoter to be turned on or off in the presence orabsence of arabinose.

Regulatable TCEs can comprise a nucleic acid sequence capable of beingbound to a binding domain of a fusion protein expressed from a regulatorconstruct such that the transcription repression domain acts to represstranscription of a nucleic acid sequence contained within theregulatable TCE.

Alternatively, the construct comprising the regulatable TCE can furthercomprise the nucleic acid sequence capable of encoding adrug-controllable (such as a drug inducible) repressor fusion proteinthat comprises a DNA binding domain and a transcription repressiondomain. In such an arrangement, the nucleic acid sequence capable ofencoding a drug-controllable (such as a drug inducible) repressor fusionprotein is on the same construct as the regulatable TCE to which therepressor fusion protein binds.

Regulatable TCEs can optionally comprise a regulator target sequence.Regulator target sequences can comprise nucleic acid sequence capable ofbeing bound to a binding domain of a fusion protein expressed from aregulator construct such that a transcription repression domain acts torepress transcription of a nucleic acid sequence contained within theregulatable TCE. Regulator target sequences can comprise one or more tetoperator sequences (tetO). The regulator target sequences can beoperably linked to other sequences, including, but not limited to, aTATA box or a GAL-4 encoding nucleic acid sequence.

The presence of a regulatable TCE and a regulator sequence, whether theyare on the same or a different construct, allows for inducible andreversible expression of the sequences operably linked to theregulatable TCE. As such, the regulatable TCE can provide a means forselectively inducing and reversing the expression of a sequence ofinterest.

Regulatable TCEs can be regulatable by, for example, tetracycline ordoxycycline. Furthermore, the TCEs can optionally comprise at least onetet operator sequence.

D. Polypeptides

Also disclosed herein are polypeptides encoded by the nucleic acidconstructs disclosed above and elsewhere herein. For example, disclosedherein are polypeptides comprising FlgM, a cleavage site, and a peptideof interest. The polypeptides can further comprise a purification tag.

In the disclosed polypeptides, the FlgM can be N-terminal to thepurification tag, the purification tag can be N-terminal to the cleavagesite, and the cleavage site can be N-terminal to the peptide ofinterest. Alternatively, the purification tag can be N-terminal to FlgM,FlgM can be N-terminal to the cleavage site, and the cleavage site canbe N-terminal to the peptide of interest. In some aspects, thepurification tag can be C-terminal to the peptide of interest.

Thus, the order of the polypeptide can be, for example, 1)Tag-FlgM-cleavage site-peptide of interest, 2) FlgM-Tag-cleavagesite-peptide of interest, 3) FlgM-cleavage site-Tag-peptide of interest,or 4) FlgM-cleavage site-peptide of interest-Tag.

1. FlgM

The disclosed polypeptides comprise FlgM. The FlgM can be wild typeFlgM. In some aspects, the FlgM can be a mutant FlgM. The mutant FlgMcan have one or more amino acid mutations compared to wild type FlgM. Insome aspects, the mutations in the mutant FlgM does not affect theability of FlgM to act as a vector for the secretion of the peptide ofinterest.

2. Cleavage Site

The disclosed polypeptides can comprise a cleavage site between FlgM andthe peptide of interest. The cleavage site can be a TEV proteasecleavage site or an ETK cleavage site. Although the cleavage site isbetween the FlgM and the peptide of interest, the cleavage site does notalways have to be contiguous with FlgM and the peptide of interest. Inother words, a purification tag can be directly before or after thecleavage site.

The cleavage site can be a protease cleavage site. The cleavage siteallows for cleavage of the polypeptide. Cleavage of the polypeptide canrelease the peptide of interest from the FlgM.

3. Peptide of Interest

The disclosed polypeptides comprise a peptide of interest. The peptideof interest can be a peptide to be expressed using the FlgM systemprovided herein.

In some aspects, the peptide of interest can be a cysteine-rich peptideor a disulfide-rich peptide. The disulfide-rich or cysteine-richpeptides can be a neuroactive toxin. The neuroactive toxin can be anyneuroactive toxin. In some aspects, the neuroactive toxin can be aconoidean derived toxin (i.e. a toxin from a conoidean). In someaspects, the neuroactive toxin can be a conopeptide. The conopeptide canbe a μ-conotoxin. Examples of μ-conotoxins include but are not limitedto SIIIA.

The peptide of interest can vary in size. In some aspects, the peptideof interest can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100 amino acids long. In some aspects, thepeptide of interest can be 5, 10, 15, 20, 25, 30, or 35 amino acidslong. The peptide of interest can vary in size. In some aspects, thepeptide of interest can be 50, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 amino acidslong. In some aspects, the peptide of interest can be 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, or 600 amino acids long.

4. Purification Tag

The disclosed polypeptides can further comprise a purification tag.Examples of purification tags include, but are not limited topoly-histidine, glutathione S-transferase (GST), Myc, HA, FLAG, andmaltose binding protein (MBP).

The purification tag can be used to purify the polypeptide after it hasbeen secreted into the culture media via the FlgM secretion system.

E. Cell Lines

Disclosed are recombinant cell lines as provided in Tables 1 and 4.

Also disclosed herein are recombinant cell lines that comprise any ofthe disclosed constructs comprising a FlgM nucleic acid sequence, acleavage site, and a nucleic acid sequence of interest. In some aspects,the recombinant cell lines can comprise one or more of the followingsequences: FlgM nucleic acid sequence, a cleavage site, and a nucleicacid sequence of interest. In some aspects, the recombinant cell linescan have a mutation of the construct comprising the FlgM nucleic acidsequence, a cleavage site, and a nucleic acid sequence of interest.

The recombinant cell lines can be derived from a wild type strain ofSalmonella enterica serovar Typhimurium. In some aspects, therecombinant cell line can be derived from a mutant strain of Salmonellaenterica serovar Typhimurium. The recombinant cell lines can be derivedfrom other enteric bacterial species. For example, the recombinant celllines can be derived from E. Coli or Yersinia.

Disclosed are recombinant cell lines, wherein the genome of therecombinant cell line comprises an alteration to one or more flagellinor hook-associated protein genes. The one or more flagellin genes can beselected from the group consisting of flgK, flgL, fliC, fljB, and fliD.

Disclosed are recombinant cell lines, wherein the cell line comprises analteration to one or more inhibitors of the flagellar FlhD4C2 masterregulatory protein complex. The inhibitors of the flagellar FlhD4C2master regulatory protein complex can be selected from the groupconsisting of fimZ, srgD, hdfR, rbsR, ompR, clpX, clpP, lrhA, ydiV,dskA, ecnR, fliT, and rcsB. Together with σ⁷⁰, FlhD4C2 activates thetranscription of class II promoters, including those of fliA, FlgM, andgenes for hook basal body assembly Inhibition of FlhD4C2 can result in areduction in FlgM or the number of hook basal body structures.Therefore, the recombinant cell lines having an alteration to one ormore inhibitors of the flagellar FlhD4C2 master regulatory proteincomplex can positively affect the secretion of disclosed polypeptides.

The recombinant cell lines can comprise a mutation to increasetranscription or translation of the FlgM T3 S-chaperone gene fliA. FliAis considered a FlgM T3 S-chaperone gene because fliA encodes for σ²⁸and σ²⁸ binds to FlgM and protects FlgM from proteolysis in thecytoplasm of the cell. Therefore, a mutation that increasestranscription or translation of the fliA gene can lead to more or betterFlgM secretion. And as disclosed in the constructs herein, FlgM is partof a polypeptide that also contains a peptide of interest. Thus, more orbetter secretion of FlgM leads to more or better secretion of thepeptide of interest. In some aspects, the mutations in the fliA generesulted in an H14N, H14D, T138I or E203D mutation in the encoded σ²⁸.

Also disclosed are combinations of any of the cells lines disclosed.These combination strains can comprise any of the disclosed constructshaving a FlgM nucleic acid sequence, a cleavage site, and a nucleic acidsequence of interest.

F. Methods

Disclosed are methods of producing a peptide of interest comprisingculturing a cell line in culture media wherein the cell line comprisesany of the disclosed polypeptides that contain FlgM, a cleavage site,and the peptide of interest. The methods can further include purifyingthe peptide of interest from the culture media.

The disclosed methods can include cell lines comprising any of thedisclosed nucleic acid constructs that contain a FlgM nucleic acidsequence, a cleavage site, and a nucleic acid sequence of interest. Thenucleic acid sequence of interest encodes the peptide of interest beingproduced.

The step of purifying the peptide of interest can include an affinitycolumn. In some aspects, the affinity column can be a σ²⁸ affinitycolumn. The affinity column can be any column designed to purify thepeptide of interest or the polypeptide of interest that contains thepeptide of interest by using an attraction between one of the peptideson the polypeptide and a molecule on the affinity column. For example, aσ²⁸ affinity column can be used because σ²⁸ binds to FlgM which is onthe polypeptide which also contains the peptide of interest. Theaffinity column can also be based on the purification tag present in thepolypeptide. In some aspects, the affinity column can have antibodiesthat bind to FlgM, the purification tag, or the peptide of interest.

The purification of the peptide of interest can include purification ofthe polypeptide that comprises FlgM, a cleavage site, and the peptide ofinterest.

The peptide of interest can be cleaved by using the cleavage sitepresent between FlgM and the peptide of interest. The peptide ofinterest can be cleaved before, after, or during purification. Forexample, using the disclosed cell lines that have a polypeptide thatincludes FlgM, a cleavage site and the peptide of interest allows forFlgM to direct the polypeptide and be secreted through a flagellar typeIII secretion system into the media that the cells are cultured in. Thepeptide of interest can be cleaved away from the rest of the polypeptideby adding a protease specific to the cleavage site of the polypeptide.The peptide of interest can then be purified from the culture media.Alternatively, the peptide of interest can be purified along with therest of the polypeptide that comprises the peptide of interest. Afterpurification, the polypeptide can be cleaved and the peptide of interestreleased. Alternatively, the peptide of interest can be cleaved duringpurification. The polypeptide can be bound to the affinity column duringpurification and while bound, the polypeptide can be cleaved releasingthe peptide of interest from the remaining polypeptide.

The cell lines of the disclosed methods can be any of the disclosedrecombinant cell lines. In some aspects, the cell lines can have aflagellar type III secretion (T3 S) system of Salmonella entericaserovar Typhimurium to secrete the polypeptide comprising the peptide ofinterest. In some aspects, the cell lines can have an alteration to oneor more flagellin genes or hook-associated protein genes. The one ormore flagellin or hook-associated protein genes can be selected from thegroup consisting of flgK, flgL, fliC, fljB, and fliD. In some aspects,the cell lines can have an alteration to one or more inhibitors of theflagellar FlhD4C2 master regulatory protein complex. The inhibitors ofthe flagellar FlhD4C2 master regulatory protein complex can be selectedfrom the group consisting of fimZ, srgD, hdfR, rbsR, ompR, clpX, clpP,lrhA, ydiV, dskA, ecnR, fliT, and rcsB. In some aspects, the cell linescan have a mutation to increase transcription or translation of the FlgMT3S-chaperone gene fliA.

G. Kits

The materials described above as well as other materials can be packagedtogether in any suitable combination as a kit useful for performing, oraiding in the performance of, the disclosed methods. It is useful if thekit components in a given kit are designed and adapted for use togetherin the disclosed method. For example disclosed are kits for producing apeptide of interest, the kit comprising one of the disclosed recombinantcell lines. The kits also can contain culture media.

The disclosed kits can also include materials for purifying the peptideof interest. For example, the kits can include an affinity column forpurifying the peptide of interest based on the purification tag presenton the polypeptide.

EXAMPLES A. Example 1 Selective Purification of Recombinant NeuroactivePeptides Using the Flagellar Type III Secretion System

In this work the flagellar FlgM protein was utilized as a vector for thesecretion of the small, highly stable pharmacologically-activepolypeptides that contain a high density of cysteine residues, whichform disulfide crosslinks in the mature product. As aproof-of-principle, a bacterial secretion system was engineered for therecombinant expression of gi-conotoxin SIIIA in Salmonella typhimurium.Using the flagellar type III secretion (T3S) apparatus, the recombinantconotoxin was selectively secreted into the culture medium, as shown inFIG. 1.

1. Materials and Methods

i. Bacterial Strains, Plasmids and Media.

Exemplary bacterial strains that can be used are listed in Table 1.Cells were cultured in Luria-Bertani (LB) media and, when necessary,supplemented with ampicillin (100 μg/ml) or tetracycline (15 μg/ml). Thegeneralized transducing phage of S. typhimurium P22 HT105/1 int-201 wasused in transductional crosses.

TABLE 1 Strains used in this study Strain Genotype LT2 Wild type TH2788fliY5221::Tn10dTc TH4885 ΔfliF5629::FKF TH5139 ΔFlgM 5628::FRT TH10874ΔFlgM 5628::FRT ΔaraBAD923::FlgM-FKF ParaBAD934 TH15360ParaBAD1036::FlgM-H6-TEV-SIIIA TH15705 fliA5225 (H14D)ΔaraBAD1034::H6-FlgM-TEV-SIIIApre TH15706 fliA5225 (H14D)ΔaraBAD1035::FlgM-TEV-SIIIApre-H6 TH15707 fliA5225 (H14D)ΔaraBAD1036::FlgM-H6-TEV-SIIIApre TH16229 ΔaraBAD1064::FlgM-DTA (Y65A)TH16240 ΔfliCD7901 ΔaraBAD1036::FlgM-H6-TEV-SIIIApre TH16778ΔprgH-hilA7791 ΔcheV7829 Δtcp-7829 ΔyhjH7740 Δaer- mcpC7834 PmotA7795ΔmotA-cheZ7888 P*flhDC7793 ΔFlgM N7753 ΔflgKL7770 Δtrg-7775 ΔycgR7775ΔmcpA7792 ΔfliB-T7771 Δtsr-7828 ecnR4::FKF Δhin- fljA7752 ΔmcpB7835ΔlrhA ΔydiV252 flgE7742::3xHA TH17020 ΔprgH-hilA7791 ΔcheV7829 Δtcp-7828ΔyhjH7740 Δaer- mcpC PmotA7795 ΔmotA-cheZ7888 P*flhD7793 ΔFlgM N7753ΔflgKL7770 Δtrg-7774 ΔycgR7775 ΔmcpA7792 ΔfljB-T7721 Δtsr-7828ΔecnR4::FKF Δhin- fljA7752 ΔmcpB7852 ΔlrhA ΔydiV flgE7742::3xHAΔaraBAD1036::FlgM-H6-TEV-SIIIApre EM170 ΔprgH-hilA7791 ΔcheV7829Δtcp-7828 ΔyhjH7740 Δaer- mcpC7834 PmotA7795 ΔmotA-cheZ7888 P*flhD7793ΔFlgM N7753 ΔflgKL7770 Δtrg-7774 ΔycgR7775 ΔmcpA7792 ΔfljB-T7721Δtsr-7828 ΔecnR4::FKF Δhin- fljA7752 ΔmcpB7852 ΔlrhA ΔydiVflgE7742::3xHA ΔaraBAD1140::FlgM-H6-TEV-MVIIA EM171 ΔprgH-hilA7791ΔcheV7829 Δtcp-7828 ΔyhjH7740 Δaer- mcpC7834 PmotA7795 ΔmotA-cheZ7888P*flhD7793 ΔFlgM N7753 ΔflgKL7770 Δtrg-7774 ΔycgR7775 ΔmcpA7792ΔfljB-T7721 Δtsr-7828 ΔecnR4::FKF Δhin- fljA7752 ΔmcpB7835 ΔlrhA ΔydiVflgE7742::3xHA ΔaraBAD1141::FlgM-H6-TEV-GV1A EM172 ΔprgH-hilA7791ΔcheV7829 Δtcp-7828 ΔyhjH7740 Δaer- mcpC7834 PmotA7795 ΔmotA-cheZ7888P*flhD7793 ΔFlgM N7753 ΔflgKL7770 Δtrg-7774 ΔycgR7775 ΔmcpA7792ΔfljB-T7721 Δtsr-7828 ΔecnR4::FKF Δhin- fljA7752 ΔmcpB7835 ΔlrhA ΔydiVflgE7742::3xHA ΔaraBAD1142::FlgM-H6-TEV-(Contulakin-G) EM173ΔprgH-hilA7791 ΔcheV7829 Δtcp-7828 ΔyhjH7740 Δaer- mcpC7834 PmotA7795ΔmotA-cheZ7888 P*flhD7793 ΔFlgM N7753 ΔflgKL7770 Δtrg-7774 ΔycgR7775ΔmcpA7792 ΔfljB-T7721 Δtsr-7828 ΔecnR4::FKF Δhin- fljA7752 ΔmcpB7835ΔlrhA ΔydiV flgE7742::3xHA ΔaraBAD1143::FlgM-H6-TEV-αVc1.1 EM174ΔprgH-hilA7791 ΔcheV7829 Δtcp-7828 ΔyhjH7740 Δaer- mcpC7834 PmotA7795ΔmotA-cheZ7888 P*flhD7793 ΔFlgM N7753 ΔflgKL7770 Δtrg-7774 ΔycgR7775ΔmcpA7792 ΔfljB-T7721 Δtsr-7828 ΔecnR4::FKF Δhin- fljA7752 ΔmcpB7835ΔlrhA ΔydiV flgE7742::3xHA ΔaraBAD1147::FlgM-H6-TEV-(Conantokin-G) EM175ΔprgH-hilA7791 ΔcheV7829 Δtcp-7828 ΔyhjH7740 Δaer- mcpC7834 PmotA7795ΔmotA-cheZ7888 P*flhD7793 ΔFlgM N7753 ΔflgKL7770 Δtrg-7774 ΔycgR7775ΔmcpA7792 ΔfljB-T7721 Δtsr-7828 ΔecnR4::FKF Δhin- fljA7752 ΔmcpB7835ΔlrhA ΔydiV flgE7742::3xHA ΔaraBAD1148::FlgM-H6-TEV-SIIIAmat EM176ΔprgH-hilA7791 ΔcheV7829 Δtcp-7828 ΔyhjH7740 Δaer- mcpC7834 PmotA7795ΔmotA-cheZ7888 P*flhD7793 ΔFlgM N7753 ΔflgKL7770 Δtrg-7774 ΔycgR7775ΔmcpA7792 ΔfljB-T7721 Δtsr-7828 ΔecnR4::FKF Δhin- fljA7752 ΔmcpB7835ΔlrhA ΔydiV flgE7742::3xHA ΔaraBAD1150::FlgM-H6-TEV-Shk EM177ΔprgH-hilA7791 ΔcheV7829 Δtcp-7828 ΔyhjH7740 Δaer- mcpC7834 PmotA7795ΔmotA-cheZ7888 P*flhD7793 ΔFlgM N7753 ΔflgKL7770 Δtrg-7774 ΔycgR7775ΔmcpA7792 ΔfljB-T7721 Δtsr-7828 ΔecnR4::FKF Δhin- fljA7752 ΔmcpB7835ΔlrhA ΔydiV flgE7742::3xHA ΔaraBAD1149::FlgM-H6-TEV-Chlorotoxin EM178ΔprgH-hilA7791 ΔcheV7829 Δtcp-7828 ΔyhjH7740 Δaer- mcpC7834 PmotA7795ΔmotA-cheZ7888 P*flhD7793 ΔFlgM N7753 ΔflgKL7770 Δtrg-7774 ΔycgR7775ΔmcpA7792 ΔfljB-T7721 Δtsr-7828 ΔecnR4::FKF Δhin- fljA7752 ΔmcpB7835ΔlrhA ΔydiV flgE7742::3xHA ΔaraBAD1144::FlgM-H6-TEV-GsMTx4 EM179ΔprgH-hilA7791 ΔcheV7829 Δtcp-7828 ΔyhjH7740 Δaer- mcpC7834 PmotA7795ΔmotA-cheZ7888 P*flhD7793 ΔFlgM N7753 ΔflgKL7770 Δtrg-7774 ΔycgR7775ΔmcpA7792 ΔfljB-T7721 Δtsr-7828 ΔecnR4::FKF Δhin- fljA7752 ΔmcpB7835ΔlrhA ΔydiV flgE7742::3xHA ΔaraBAD1145::FlgM-H6-TEV-Calciseptine

ii. Construction of Chromosomally Expressed FlgM-Toxin Fusions.

SIIIA was amplified in a de novo fill-in PCR-reaction using a longprimer SIIIA_long_fw(CAGAACTGCTGCAACGGCGGCTGCAGCAGCAAATGGTGCCGCGATCATGCG CGCTGCTGCGGCCGC;SEQ ID NO: 1) covering all 66 base pairs encoding for SIIIA(QNCCNGGCSSKWCRDHARCCGR; SEQ ID NO:2). The SIIIA sequence was designedaccording to the optimal codon usage of Salmonella typhimurium. The 66bp sequence was duplexed with the help of a short reverse primer(SIIIA_short_rv: GCGGCCGCAGCAGCGCGCATG; SEQ ID NO:3) initiating thefill-in from the 3′ end of the template primer.

A cleavage site for the TEV protease (ENLYFQG; SEQ ID NO:4) and apoly-histidine tag (H6 encoded by (CATCAC)₃; SEQ ID NO:5) were insertedduring amplification of flgM and SIIIA at various positions, resultingin three different construct (named construct 1-3).

In the following, the construction procedure of construct 1 will beexplained exemplarily in more detail, Construct 2 and construct 3 weredesigned accordingly (see Table 2 for primer sequences). The flgM genewas amplified from genomic DNA (TH2788) using forward primersI_HS1_FlgM_fw and reverse primers I_HS2_FlgM_rv. The reverse primersencoded for an 18 bp overhang that was homologous to the 5′ SIIIAsequence (see above). To increase the length of the homologous region,SIIIA was amplified from the previous synthesized template with primers1_HS3_SIIIA_fw and 1_HS4_SIIIA_rv-adding an additional 10 bp overlapwith homology to the sequence of the TEV protease cleavage site. The PCRproducts of FlgM and SIIIA were purified and used in a subsequent fusionPCR as a template together with forward primers 1_HS1_FlgM_fw andreverse primers 1_HS4_SIIIA_rv. This method allows the fusion of two PCRproducts that share a homology of (in case of construct 1) 28 basepairs, resulting in one long flgM-SIIIA fusion construct. All constructscontained a 5′-BamH1 and a 3′-EcoR1 restriction site for cloning intopUC18, resulting in the subcloning vectors (pHS1 (pUC18BamHI-His6-FlgM-TEV-SIIIA-EcoRI), pHS2 (pUC18BamHI-FlgM-TEV-SIIIA-His6-EcoRI) and pHS3 (pUC18BamHI-FlgM-His6-TEV-SIIIA-EcoRI).

TABLE 2 Primer sequences for toxin construction. Primer name SequenceSIIIA_long_fw CAGAACTGCTGCAACGGCGGCTGCAGCAGCAAATGGTGCCGCGATCATGCGCGCTGCTGCGGCCGC (SEQ ID NO: 1) SIIIA_short_rvCGGCCGCAGCAGCGCGCATG (SEQ ID NO: 3) 1_HS1_FlgM_fwcgggatcccgATGCATCACCATCACCATCACATGAGCATTGA CCGTACCTC (SEQ ID NO: 6)1_HS2_FlgM_rv CCGTTGCAGCAGTTCTGgccctgaaaatacaggttttcTTTACTCTGTAAGTAGCTCTG(SEQ ID NO: 7) 1_HS3_SIIIA_fwttttcagggcCAGAACTGCTGCAACGGCGG (SEQ ID NO: 8) 1_HS4_SIIIA_rvggaattccTTAGCGGCCGCAGCAGCGC (SEQ ID NO: 9) DTA-FlgM_fwACTCGCTCATTCGCGAGGCGCAGAGCTACTTACAGAGTAAAGGCAGCTCTCACCACCACC (SEQ ID NO: 10) DTA-FlgM_rvTTCATCAACGCGCCCCCCATGGGACGCGTTTTTAGAGGCATTAACGGTTACCTGCACAAG (SEQ ID NO: 11) 2_HS5_FlgM_fwcgggatcccgATGAGCATTGACCGTACCTTC (SEQ ID NO: 12) 2_HS6_FlgM_rvCCGTTGCAGCAGTTCTGgccctgaaaatacaggttttcTTTACTCTGTAAGTAGCTCTGC (SEQ ID NO: 13) 2_HS7_SIIIA_fwttcagggcCAGAACTGCTGCAACGGC (SEQ ID NO: 14) 2_HS8_SIIIA_rvggaattccTTAGTGATGGTGATGGTGATGGCGGCCGCAGCA GCGCGCAT (SEQ ID NO: 15)3_HS9_FlgM_rv gccctgaaaatacaggtatcGTGATGGTGATGGTGATGTTTACTCTGTAAGTAGCTCTG (SEQ ID NO: 16) 3_HIS10_SIIIA_fwTCACgaaaacctgtattacagggcCAGAACTGCTGCAACGGCGGC (SEQ ID NO: 17)3_HIS11_SIIIA_rv ggaattccTTAGCGGCCGCAGCAGCGCG (SEQ ID NO: 18)1A-MVIIA_fw TGCAAAGGTAAAGGTGCAAAATGTAGCCGTCTGATGTATGATTGTTGTACCGGTAGCTGT (SEQ ID NO: 19) 1B-MVIIA_rvTTAACATTTACCGCTACGACAGCTACCGGTACAACAAT (SEQ ID NO: 20)1C-MVIIA_homology_fw ACATCACCATCACCATCACgaaaacctgtatcagggcTGCAAAGGTAAAGGTGCAAA (SEQ ID NO: 21) 1D-MVIIA_homology_rvttcatcaacgcgccccccatgggacgcgtttttagaggcaTTAACATTTACCGCTACGAC (SEQ ID NO: 22) 2A-GVIA_fw TGTAAAAGTCCGGGTAGCAGCTGTAGCCCGACCAGCTATAATTGTTGTCGTAGCTGTAAT (SEQ ID NO: 23) 2B-GVIA_rvTTAATAGCAACGTTTGGTATACGGATTACAGCTACGAC AACAAT (SEQ ID NO: 24)2C-GVIA_homology_fw ACATCACCATCACCATCACgaaaacctgtattttcagggcTGTAAAAGTCCGGGTAGCAG (SEQ ID NO: 25) 2D-GVIA_homology_rvttcatcaacgcgccccccatgggacgcgtttttagaggcaTTAATAGCAACGTTTGGTAT (SEQ ID NO: 26) 3A-Contulakin-G_fwGAAAGCGAAGAAGGTGGTAGCAACGCAACCAAAAAAC CGTATATTCTGTAA (SEQ ID NO: 27)3B-Contulakin-G_rv TTACAGAATATACGGTTTTTTGGTTGCGTTGCTACCACCTTCTTCGCTTTC (SEQ ID NO: 28) 3C-Contulakin-G_ACATCACCATCACCATCACgaaaacctgtatatcagggcGAAAGC homology_fwGAAGAAGGTGGTAG (SEQ ID NO: 29) 3D-Contulakin-G_ttcatcaacgcgccccccatgggacgcgtattagaggcaTTACAGAATATACG homology_rvGTTTTT (SEQ ID NO: 30) 4A-alpha-Vc1.1_fwTGTTGTAGCGATCCGCGTTGTAATTATGATCATCCGGAA ATTTGCTAA (SEQ ID NO: 31)4B-alpha-Vc1.1_rv TTAGCAAATTTCCGGATGATCATAATTACAACGCGGATCGCTACAACA (SEQ ID NO: 32) 4C-alpha-Vc1.1_ACATCACCATCACCATCACgaaaacctgtattttcagggcTGTTGTA homology_fwGCGATCCGCGTTG (SEQ ID NO: 33) 4D-alpha-Vc1.1_ttcatcaacgcgccccccatgggacgcgtattagaggcaTTAGCAAATTTCCG homology_rvGATGAT (SEQ ID NO: 34) 9A-GsMTx4_fwTGTCTGGAATTTTGGTGGAAATGCAATCCGAACGATGATAAATTGTTGTGTTCCGAAACTG (SEQ ID NO: 35) 9B-GsMTx4_rvACCGCTGCTAAAATTGCACAGTTTAAACAGTTTGCTGCATTTCAGTTTCGGACGACAACA SEQ ID NO: 36) 9C-GsMTx4_homology_fwACATCACCATCACCATTCACgaaaacctgtattttcagggaGTCTGGAATTTTGGTGGAA (SEQ ID NO: 37) 9D-GsMTx4_homology_rvatcaacgcgccccccatgggacgcgtttttagaggcaTTAACCGCTGCTAAAATTGCACA (SEQ ID NO: 38) 10Aa-Calciseptine_fwCGCATCTGCTATATTCATAAAGCAAGCCTGCCTCGTGCAACCAAAACCTGTGTTGAAAAT (SEQ ID NO: 39) 10ab-Calciseptine_rvTATATTCGCGCTGGGTACGAATAAACATTTTATAGCAGGTATTTTCAACACAGGTTTTGG (SEQ ID NO: 40) 10Ba-Calciseptine_fwTCGTACCCAGCGCGAATATATCAGCGAACGTGGTTGTGGTTGTCCGACCGCAATGTGGCC (SEQ ID NO: 41) 10Bb-Calciseptine_rvTTTGTTGCAACGATCACCTTTACAACATTCGGTCTGATACGGCCACATTGCGGTCGGACA (SEQ ID NO: 42) 10C-ACATCACCATCACCATCACgaaaacctgtaancagggcCGCATCT Calciseptine_homology_fwGCTATATTCATAA (SEQ ID NO: 43) 10D-atcaacgcgccccccatgggacgcgtttttagaggcaTTATTTGTTGCAACGCalciseptine_homology_rv ATCACCTT (SEQ ID NO: 44) 5A-Conantokin-G_fwGGTGAAGAAGAACTGCAAGAAAACCAAGAACTGATTC GCGAAAAAAGCAATTAA (SEQ ID NO: 45)5B-Conantokin-G_rv TTAATTGCTTTTTTCGCGAATCAGTTCTTGGTTTTCTTGCAGTTCTTCTTCACC (SEQ ID NO: 46) 5C-Conantokin-G_ACATCACCATCACCATCACgaaaacctgtattttcagggcGGTGAAG homology_fwAAGAACTGCAAGA (SEQ ID NO: 47) 5D-Conantokin-G_ttcatcaacgcgccccccatgggacgcgtttttagaggcaTTAATTGCTTTTTTC homology_rvGCGAA (SEQ ID NO: 48) 6A-mu-SIIIA_fwGAAAATTGCTGTAATGGTGGTTGTAGCAGCAAATGGTGTCGTGATCATGCACGTTGTTGT (SEQ ID NO: 49) 6B-mu-SIIIA_rvTTAACAACAACGTGCATGATCACGACACCATTTGCTGCTACAACCACCATTACAGCAATT (SEQ ID NO: 50) 6C-mu-SIIIA_homology_fwACATCACCATCACCATCACgaaaacctgtattttcagggeGAAAATTGCTGTAATGGTGG (SEQ ID NO: 51) 6D-mu-SIIIA_homology_rvttcatcaacgcgccccccatgggacgcgtttttagaggcaTTAACAACAACGTGCATGAT (SEQ ID NO: 52) 7A-Shk_fw AGCTGCATTGATACCATFCCGAAAAGCCGTTGTACCGCATTTCAGTGTAAACACAGCATG (SEQ ID NO: 53) 7B-Shk_rvACAGGTGCCACAGGTTTTACGACAAAAGCTCAGACGATATTTTCATGCTGTGTTTACACTG (SEQ ID NO: 54) 7C-Shk_homology_fwATCACCATCACCATCACgaaaacctgtacagggcCGTAGCTGCATTGATACCATTC (SEQ ID NO: 55) 7D-Shk_homology_rvcatcaacgcgccccccatgggacgcgtttttagaggcaTTAACAGGTGCCACAGGTTTTA (SEQ ID NO: 56) 8A-Chlorotoxin_fwATGTGTATGCCGTGTTTTACCACCGATCATCAGATGGCACGTAAATGTCATGATTGTTGT (SEQ ID NO: 57) 8B-Chlorotoxin_rvGACACTGCGGACCATAACATTTACCGCGACCTTTACCACCACAACAATCATCACATTTAC (SEQ ID NO: 58) 8Bb-Chlorotoxin_rvttaACGACACAGACACTGCGGACCATAACAT (SEQ ID NO: 59)8C-Chlorotoxin_homology_fw ACATCACCATCACCATCACgaaaaccacagggcATGTGTATGCCGTGTTTT (SEQ ID NO: 60) 8D-Chlorotoxin_homology_rvttcatcaacgcgccccccatgggacgcgtttttagaggcattaACGACACAGACACTGCG (SEQ ID NO: 61)

All flgM-SIIIA fusions were amplified from the respective subcloningvectors with primers having homologous regions for the native flgM locusor the arabinose locus (ΔaraBAD), respectively. Chromosomal insertionswere constructed using X-Red mediated recombination.

The expression of SIIIA in Salmonella resulted in a recombinant peptidethat was lacking posttranslational modifications that are usuallypresent under physiological conditions. Those include a neutralN-terminal pyroglutamate, C-terminal amidation and cleavage of atwo-residue fragment—named SIIIApre. However, according to functionalstudies on the related β-conotoxin GIIIA, the potency of SIIIA isdetermined by the lysine at position 11 and therefore not affected byany N- and C-terminal sequence variations.

All other toxins from various organisms (snails, spiders, snakes, andsea anemone) were constructed correspondingly, and their sequences arelisted in Table 3 and Table 2.

TABLE 3 Toxins used in this study. Toxin size Amino acid Toxin nameOrganism Species [aa] sequence w/o modifications) ω-MVIIA Cone Conus  25CKGKGAKCSRLMYDCCTGSCR snail magus SGKC (SEQ ID NO: 62) ω-GVIA Cone Conus 27 CKSPGSSCSPTSYNCCRSCNPY snail geographus TKRCY (SEQ ID NO: 63)Contulakin-G Cone Conus  16 ESEEGGSNATKKPYIL snail geographus(SEQ ID NO: 64) α-Vc1.1 Cone Conus  16 GCCSDPRCNYDHPEIC snail victoriac(SEQ ID NO: 65) Conantokin-G Cone Conus  17 GEEELQENQELIREKSN snailgeographus (SEQ ID NO: 66) μ-SIIIAmat Cone Conus  20ENCCNGGCSSKWCRDHARCC snail sirlanis (SEQ ID NO: 67) μ-SIIIApre ConeConus  22 QNCCNGGCSSKWCRDHARCC snail sirloins GR (SEQ ID NO: 68) ShkSea  Stichodactyla  35 RSCIDTIPKSRCTAFQCKHSMK anemone helianthusYRLSFCRKTCGTC (SEQ ID NO: 69) Chlorotoxin Scorpion Leiurus  36MCMPCFTTDHQMARKCDDCC quinquestriatus GGKGRGKCYGPQCLCR (SEQ ID NO: 70)GsMTx4 Spider Grammostola  35 GCLEFWWKCNPNDDKCCRPK spatulataLKCSKLFKLCNFSSG (SEQ ID NO: 71) Calciseptine Snake Dendroaspis  60RICYIHKASLPRATKTCVENTC p. polylepis YKMFIRTQREYISERGCGCPTAMWPYQTECCKGDRCNK (SEQ ID NO: 72) DTA Y65A Bacterium Corynebacterium 190GADDVVDSSKSFVMENFSSYH diphtheriae  GTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAA GASVDNENPLSGKAGGVVKVT YPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFG DGASRVVLSLPFAEGSSSVEYIN NWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNR (SEQ ID NO: 73)

Fragment A of the diphtheria toxin (DTA Y65A) was amplified from plasmidpTH794 with primers DTA-FlgM_fw(ACTCGCTCATTCGCGAGGCGCAGAGCTACTTACAGAGTAAAGGCAGCTCTC ACCACCACC; SEQ IDNO:10) and DTA-FlgM_rv (TTCATCAACGCGCCCCCCATGGGACGCGTTTTTAGAGGCATTAACGGTTACCTGCACAAG; SEQ ID NO: 11). DTA amplification included apoly-histidine-tag (H6) with a GSS-linker before the His-tag and aSSGLVPR-linker between the His-tag and the first amino acid of DTA. DTAwas chromosomally inserted by 2-Red-mediated recombination. Theinsertion was carried out in a ΔaraBAD::FlgM⁺ background. DTA wastargeted in front of the flgM stop codon resulting in a translationalFlgM-DTA fusion.

iii. Recombinant Expression and Purification of SIIIA Conotoxin.

Strains expressing SIIIA conotoxin fusions were picked from a freshsingle colony and grown in 10 ml LB overnight. The overnight cultureswere diluted 1:100 into 1 l fresh media and grown for six hours. Ifappropriate, SIIIA expression was induced after the first two hours byaddition of 0.2% arabinose. Cells were pelleted by centrifugation (7,000rpm), and the supernatant containing FlgM-SIIIA was passed through a0.22 μm polyethersulfone filter (Corning, N.Y., USA)—a low proteinbinding membrane for removal of residual bacteria. For furtherpurification, a gravity-flow column (Bio-Rad) packed with 3 g Ni-IDAresin (Protino Ni-IDA, Machery-Nagel) was used and affinity taggedproteins were eluted under native conditions at pH 7.5 with a buffercontaining 250 mM imidazole.

iv. Secretion Assay.

Overnight cultures were diluted 1:100 in LB and grown for two hours at37° C. before inducing the expression of the respective FlgM-toxinfusion by adding 0.2% L-arabinose. Cells were kept at 37° C. for anadditional four hours, while the fusion proteins were expressed. After atotal of six hours, the optical density (OD) at 600 nm was determinedfor all strains.

Two ml aliquots of the resulting cell culture were centrifuged for 10min at 4° C. and 7,000 rpm to obtain, for each aliquot, a pellet andsupernatant. The supernatant was filtered through a low protein bindingfilter with 0.2 μm pore size (Acrodisk Syringe Filter, PALL LifeSciences) to remove remaining cells. Alternatively, aliquots werecentrifuged twice at maximum speed to remove residual cells. Secretedproteins in the filtered or twice-centrifuged supernatant wereprecipitated by addition of TCA (10%0/final concentration). Thesupernatant samples were resuspended in 2×SDS sample buffer (100 mM TrispH 6.8, 4% SDS, 10% glycerol, 2% β-mercaptoethanol, 25 mM EDTA, 0.04%bromophenol blue) and adjusted to 20 OD₆₀₀ units per μl. The cellular,pellet fraction was suspended in 2×SDS sample buffer, whose volume wasadjusted to yield 20 OD₆₀₀ units per μl.

v. SDS PAGE and Western Blotting.

Expressed FlgM-toxin fusions of whole-cell lysate and culturalsupernatant were subjected to SDS polyacrylamide gel electrophoresis andanalyzed by immunoblotting using polyclonal anti-FlgM and polyclonalanti-FliK antibodies (rabbit) for detection. Antigen-antibody complexeswere visualized by chemiluminescent or infrared detection using theLI-COR Odyssey imaging system. For chemiluminescent development,secondary goat α-rabbit antibodies (Bio-Rad) conjugated with horseradishperoxidase (HRP) and an ECL detection kit (Amersham Biosciences) wereused. For infrared detection, secondary anti-rabbit-IRDye690 (LI-COR)was used. Densitometric measurements of FlgM-SIIIA and FliK bands wereperformed using ImageJ 1.45s for Mac OS X.

vi. TEV Protease Cleavage.

SIIIA was cleaved off flagellar secretion substrates using AcTEVProtease (Invitrogen). 450 U of TEV protease was added to 15 ml of theelution fraction, which corresponds to the secreted proteins from a 750ml culture. Cleavage was performed overnight at room temperature inelution buffer containing 1 mM dithiothreitol (DTT) and 0.5 mM EDTA.

vii. Folding of SIIIA.

Recombinant SIIIA was folded in 0.1 M Tris buffer, pH 7.5 using 1 mMoxidized glutathione (GSSG) and 0.1 mM EDTA. The folding reaction wasallowed to proceed overnight at room temperature and was quenched by theaddition of formic acid to a final concentration of 8%.

viii. Solid Phase Extraction of SIIIA.

C₁₈ Solid phase extraction columns (Supelclean LC-18, Supelco) were usedto remove the oxidized peptide from the other components of the foldingand cleavage reactions. The isolated peptide was then dried and purifiedby HPLC on a Waters 600 chromatograph, equipped with a dual-wavelengthabsorbance detector and a Vydac C₁₈ analytical column. The HPLCseparation was run with a linear gradient from 4.5% to 31.5% (aq)acetonitrile (0.9% change per minute), maintaining 0.1% TFA throughout.This allowed purification of the correct folding product, mass wasvalidated by matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF) mass spectrometry.

ix. Electrophysiology of Mammalian Nay Channels.

The functional activity of recombinant SIIIA was assessedelectrophysiologically with Xenopus oocytes expressing Na_(V)1.2.Briefly, the oocyte was placed in a 30 μl recording chamber perfusedwith ND96 and two-electrode voltage clamped. A holding potential of −80mV was used, and activation of sodium channels was achieved by steppingthe potential to −10 mV for 50 ms every 20 seconds. Exposure to toxinwas performed in a static bath to conserve peptide, as follows. Prior totoxin application, the perfusion of the bath was halted, and controlresponses were recorded. Peptide (3 μl at 10-times the finalconcentration) was then applied to the bath, which was stirred for 5seconds using a micropipette. Recordings of the onset of block by thepeptide were obtained for about 20-min, after which the perfusion withND96 was resumed to remove unbound peptide while the rate of recoveryfrom block was monitored for about 20 minutes, The time course of theonset of block was fit to a single-exponential function to obtain theobserved rate constant of block, k_(obs). The rate of recovery fromblock during peptide washout was too slow to measure by curve fitting,so k_(off) was estimated from the level of recovery after 20 min ofwashing and assuming exponential decay of block. All recordings wereobtained at room temperature.

x. Immunostaining.

Fluorescent microscopy analysis was performed.

2. Results and Discussion.

Conotoxins are synthesized in the venom duct of marine cone snails andtarget ion channels, including voltage-gated sodium channels (VGSCs).SIIIA is a μ-conotoxin from Cornus striatus that inhibits tetrodotoxin(TTX)-resistant VGSCs in frog and TTX-sensitive VGSCs in rodents. Thei-Conotoxin SIIIA binds to site 1 of the α-subunit of VGSCs and therebyoccludes the channel pore and blocks sodium conductance. Underphysiological conditions two forms of SIIIA exist: a precursor form anda mature peptide, which has been modified by processing the C-terminalglycine-arginine residues to an amino group and changing the N-terminalglutamine residue to pyroglutamate. Herein, two forms of SIIIA wererecombinantly expressed, the precursor peptide and a peptide that mostclosely resembles the physiological mature form (N-terminal glutamateinstead of pyroglutamate). In mammalian preparations, i-SIIIAeffectively blocks neuronal sodium channels such as Na_(v)1.2 and 1.6,and the skeletal muscle subtype Na 1.4. Because SIIIA targets VGSCs andhas potent analgesic activity in mice, it can be used for medicalresearch and drug discovery. The fundamental advantage in developing aT3SS bacterial expression system is that after translation thepolypeptides translocate through a narrow secretion channel in theflagellar structure and thereby bypass the problem of aggregation ininclusion bodies. Polypeptide secretion prevents intermolecularaggregation in the cytoplasm. Intramolecular disulfide bonds form as thepolypeptide chain exits the reductive environment of the bacterialcytoplasm and enters an oxidizing extracellular environment.

The efficiency with which different constructs serve as type IIIsecretion substrates was assessed. The SIIIA pretoxin (“μ-SIIIApre” inTable 3 and henceforth referred to as “rSIIIA”) is a small 22 amino acidpeptide which was fused to FlgM with an N-terminal TEV cleavage site infront of SIIIA. In order to ensure proper secretion and to diminish thepossibility of interference with the structural secretion signal ofFlgM, three different constructs were designed with a poly-histidine tagat different positions. All constructs were chromosomally expressed fromthe arabinose-inducible promoter P_(araBAD) FIG. 1B shows the secretionprofile of all three constructs. Construct 3 (FlgM-H6-TEV-SIIIA)displayed the most prominent secretion product; construct 2(FlgM-TEV-SIIIA-H6) exhibited the lowest secretion level. This secretionranking was consistent in all backgrounds tested, some of which carrieda mutation (fla* or H14D) in the σ²⁸ structural gene fliA that enhancesstability of this FlgM secretion-chaperone protein. FlgM levels werevery low in a strain unable to form a flagellar structure (ΔfliF)compared to those in a wild type strain.

The flagellum-specific transcription factor σ²⁸ acts as a chaperone tofacilitate FlgM secretion. As shown in Figure Cc, σ²⁸ H14D, with itsenhanced stability, increased levels of intracellular and secretedFlgM-SIIIA. σ²⁸ H41D also increases FlgM stability. This is the cause ofthe increased levels of FlgM-SIIIA in the fliA* background (FIG. 1C,lanes 2 and 3). The effect of removing the late flagellar secretionsubstrates (and potential secretion competitors) FliC and FliD (ΔfliCD)was also tested. The fliC and fliD genes are divergently transcribed.However, the fliD gene is in an operon with fliT, which encodes the FliTinhibitor of all flagellar gene expression. Thus, the ΔfliCD deletionresults in removal of secretion competitors for FlgM in addition toincreasing the number of flagellar basal structures. The net effect wasincreased production and secretion of both FlgM and FlgM-SIIIA from thecell (FIG. 1C, lanes 2 and 4).

As FlgM-H6-TEV-SIIIA showed the highest level of secretion, thisconstruct was used for further purification experiments. The SIIIAfusion was expressed from an arabinose inducible promoter to allowsynchronization of the high level of expression with completion of theflagellar secretion systems. Secreted FlgM-H6-TEV-SIIIA was separatedfrom the bacterial cell culture by centrifugation and was obtained fromthe supernatant under native conditions by nickel-affinitychromatography. As shown in FIG. 2A, FlgM-H6-TEV-SIIIA efficiently boundto the Ni-IDA resins and was eluted with imidazole-containing elutionbuffer. After concentration of the eluted fraction, rSIIIA was cleavedfrom the FlgM-H6-TEV secretion signal using TEV protease. Western Blotanalysis and Coomassie staining demonstrated that TEV cleavage wascomplete after 3 hours at room temperature.

As with all cysteine-rich peptides, the formation of the nativedisulfide bonds is a process involved in generating biologically activeSIIIA. However, the optimal buffer conditions of the TEV protease can be1 mM dithiothreitol (DTT). This redox agent provides reducing power forthe TEV protease but at the same time can reduce S—S bonds in thepeptide of interest. For this reason, rSIIIA was refolded prior toelectrophysiological analysis. Refolding was carried out in a redoxbuffer of oxidized and reduced glutathione, allowing thiol-disulfideexchange reactions under conditions that are known to favor formation ofthe native disulfide bonds.

Tests of the rSIIIA precursor form on Xenopus oocytes expressing ratNa_(V)1.2 demonstrated that its functional activity was similar to thatof chemically synthesized SIIIA. Sodium currents in response todepolarization were recorded. FIG. 2B presents the results from arepresentative experiment in which rSIIIA blocked Na_(V)1.2 conductance[compare the peak current of the oocyte before (gray trace) and afterrSIIIA exposure (black trace)]. The kinetics of the onset of blockfollowing exposure to 10 μM rSIIIA and the recovery from block followingits washout were assessed. The observed rate constant for the onset ofblock (k_(obs)) and the rate constant for recovery from block (k_(off))were, respectively, 0.17±0.04 min⁻¹ and 0.0043±0.0004 min⁻¹ (mean±SD,n=3 oocytes). The k_(off) value is close to that observed withchemically synthesized SIIIA; however, the k_(obs) value is aboutsix-fold lower than that observed with chemically synthesized SIIIA. Thedifference in k_(obs) is due to the disparity in the sequences of thetwo peptides (N-terminal glycine carryover after TEV cleavage andlacking post-translational modifications).

In addition to the secretion of SIIIA pretoxin, several other toxinsfrom various organisms, such as spider (GsMTx4), scorpion (Chlorotoxin),snake (Calciseptine), snails (ω-MVIIA, ω-GVIA, Contulakin-G, α-Vc1.1,Conantokin-G, mature SIIIA) and sea anemone (Shk) were tested forrecombinant expression and subsequent secretion in a complementaryapproach (see Table 3 for a detailed list). Although there weredifferences in cellular levels and secretion efficiencies, the resultsshown in FIG. 2C demonstrate that all tested toxins were secreted intothe culture supernatant. The secretion of the unstable 190 amino acidlong catalytic subunit of diphtheria toxin fused to FlgM was also tested(FIG. 2D). The proteolytic degradation that was observed within thecytosolic fraction did not occur once the protein was secreted from thecell.

For further optimization of the Salmonella secretion strain, anon-motile poly-hook mutant was constructed that had the followingcharacteristics: It lacked the flagellum-related SPI-1 (ΔprgH-hilA,)virulence system, as well as all class 3 flagellar genes (ΔflgKL,ΔfljB-T, ΔFlgM N, Δaer-mcpC, PmotA, ΔmotA-cheZ, ΔmcpA, ΔmcpB, Δtsr) thatcould diminish or interfere with flagellar type III secretion of thetoxin fusion of interest. In addition, the strain harbored a mutation inthe flhD promoter (P*flhD) and was deleted for negative regulators ofthe flagellar master regulator FlhD4C2 (ΔlrhA, ΔydiV, ΔecnR). LrhA actsas a negative regulator of flhDC transcription by directly binding tothe promoter region of the master regulator. The EAL domain protein YdiVposttranslationally regulates the activity of FlhD4C2 protein bytargeting the complex for ClpXP-dependent degradation and therebynegatively influences FlhDC-dependent flagellar class 2 promoteractivity. Transposon insertions in those negative regulators of FlhDCincreases flagellar gene expression.

As shown by fluorescent labeling of the flagellar hook structures inFIG. 3A, these mutations resulted in a roughly two-fold increase offlagellar secretion structures per cell compared to wild type cells,which in turn led to a substantial increase in secretion rate offlagellar proteins. Secretion rates of FliK and FlgM-SIIIA (construct 3)were roughly two-fold higher in the poly-hook background compared tothose in the wildtype and fliA* backgrounds (FIG. 3B).

A major advantage of this system is the workflow. After induction witharabinose, the bacterial cells express recombinant polypeptides andsubsequently secrete the toxin into the media. Contrary to thepurification of neuropeptide by traditional methods, this makes itunnecessary to lyse cells in order to purify toxins. Instead,recombinant SIIIA was selectively secreted via the flagellar T3 SS andit accumulated in the culture supernatant. After several hours ofproduction, cells were removed by simple centrifugation and filtration.Other aspects of this system are type III secretion chaperones thatfacilitate secretion or increase the stability of the secretionsubstrates. Type III secretion substrates are usually secreted in anunfolded or partially folded state, which is often achieved byinteraction of the chaperones with the secondary structure of thesecreted protein. Since SIIIA was fused to FlgM, a type III secretionsubstrate, it was protected from premature folding by interaction withFliA (σ²⁸), the cognate type III secretion chaperone of FlgM. For thedisclosed recombinant expression, full-length FlgM was used, whichincreased secretion efficiency due to the presence of its FliA chaperonebinding site.

The disclosed system provides an easy-to-use method for complicatedtoxin purification a task that goes beyond yield and solubility ofpeptides, but also allows the peptides and proteins to be accessible ina correctly folded and active state.

B. Example 2 Analysis of Factors that Affect Salmonella FlgM ProteinSecretion

In this work, a new approach was initiated that uses an expressionsystem that bypasses the inclusion body problem of recombinant smallpeptide expression. It exploits the flagellar secretion system ofSalmonella enterica serovar Typhimurium (Salmonella typhimurium) thathas been shown to export non-flagellar proteins if fused to flagellarsecretion signals, e.g. hook protein FlgE or flagellin FliC. Theflagellar secretion system is a member of a family of bacterial type IIIsecretion systems that selectively secrete proteins from the cytoplasmto the external medium. Almost all type III secretion systemscharacterized thus far are either required for the secretion ofvirulence determinants for a number of plant and animal pathogens or forthe secretion of proteins required for the structure and function of thebacterial flagellum. The bacterial flagellum is composed of an externalhelical filament, extending many body lengths from the cell surface,attached to a rotary motor embedded within the cell wall and membranes.For Salmonella, a chemosensory system controls the clockwise andcounterclockwise direction of flagellar rotation, which allows thebacterium to migrate towards attractants, such as nutrients, or awayfrom repellents that are harmful to the bacterium.

The specificity for flagellar substrate secretion is primarilydetermined by an N-terminal peptide secretion signal that has adisordered structure. Many secretion substrates also require a specificsecretion chaperone to facilitate secretion. One flagellar secretionsubstrate, FlgM, is a regulatory protein that couples flagellar assemblyto gene expression. σ²⁸, a transcription factor specific for flagellarpromoters, directs transcription of genes specifically needed aftercompletion of the flagellar motor and thereby induces expression of thefilament and chemosensory genes. FlgM binds σ²⁸ and prevents itsassociation with RNA polymerase. Completion of the flagellar motorresults in a change in substrate specificity of the flagellar secretionapparatus, switching from secretion of proteins required for motorassembly to late substrate secretion of proteins needed in filamentassembly. FlgM itself is a late secretion substrate and its secretionreleases σ²⁸, enabling it to transcribe the filament and chemosensorygenes only upon completion of the motor. At the same time, the α²⁸transcription factor acts as the secretion chaperone that facilitatesthe secretion of its inhibitor, FlgM.

In this work the flagellar FlgM protein was utilized as a vector for thesecretion of the small, highly stable pharmacologically-activepolypeptides that contain a high density of cysteine residues, whichform disulfide crosslinks in the mature product. A bacterial secretionsystem was created for the recombinant expression of μ-conotoxin SIIIAin Salmonella typhimurium. Using the flagellar type III secretion (T3S)apparatus, the recombinant conotoxin was selectively secreted into theculture medium.

1. Materials and Methods

i. Bacteria Strains and Growth Conditions.

All strains used in this study are derivatives of LT2, a wild-typestrain Salmonella enterica serovar Typhimurium, and are listed in Table4. All strains were constructed during the course of this study. Strainconstruction utilized either P22-mediated generalized transduction or λRed-mediated targeted mutagenesis. For strains with the flhDC operonexpressed from its native promoter, flagellar gene expression wasinduced by 100-fold dilution from overnight stationary cultures intofresh LB medium (10 g Bacto tryptone, 5 g Bacto Yeast Extract and 5 gNaCl per liter). For strains with the flhDC operon expressed from thetetA promoter of transposon Tn10 (ΔP_(flhDC)8089::tetR-PtetA), flagellargene expression was induced by addition of the tetracycline analoganhydro-tetracycline (1 μg/ml). For all strains used in this study, thearabinose utilization operon araBAD, was replaced with the FlgM⁺ gene orFlgM gene fusions FlgM-6His-TEV-δ-SVIE or FlgM-6His-ETK-δ-SVIE(ΔaraBAD::FlgM⁺, ΔaraBAD::FlgM-6His-TEV-δ-SVIE orΔaraBAD::FlgM-6His-ETK-δ-SVIEI, respectively). This allowed for theinduction of FlgM, FlgM-6His-TEV-δ-SVIE and FlgM-6His-ETK-δ-SVIEproduction from the P_(araBAD) promoter by the addition of L-arabinoseto the growth medium. Arabinose was added to 0.2% final concentrationtwo hours after the induction of the flhDC operon. After another 5hours, the cultures were centrifuged at 10,000 g for 30 min to pelletthe cells. The supernatant was filtered with 0.2 um low protein bindingfilter (Acrodisk Syringe Filter, PALL Life Sciences) to remove remainingcells. Secreted proteins in the filtered supernatant were precipitatedby addition of TCA (trichloroacetic acid) to 10% final concentration.The cell pellet was re-suspended in cold PBS (phosphate buffered saline)containing 5 mM PMSF (phenylmethylsulfonyl fluoride), followed bysonification to lyse the cell suspension. The cell lysate was eitheranalyzed directly for whole cell protein or separated into soluble andinsoluble fractions analysis by 30 min centrifugation (15,000 g) at 4°C. To test the effect of different concentration of NaCl and KCl on FlgMsecretion, LB medium was prepared without NaCl and either NaCl or KClwere added to the desired concentrations.

TABLE 4 Strains and derivatives of LT2. Strain Genotype TH17831ΔaraBAD1124::FlgM-6His-TEV-δ-SVIE TH18353ΔaraBAD1124::FlgM-6His-TEV-δ-SVIE DclpX::tetRA TH18500ΔaraBAD1156::FlgM⁺ TH18527 ΔaraBAD1156::FlgM⁺ ΔclpX::tetRA TH18528ΔaraBAD1156::FlgM⁺ ΔflhDC::FKF TH18549 ΔaraBAD1156::FlgM⁺ ΔflhDC::FKFΔclpX::tetRA TH18624 ΔaraBAD1156::FlgM⁺ ΔfliA5805::tetRA TH18636ΔaraBAD1156::FlgM⁺ fliA8088(H14D, R91C, L207P) TH18647ΔaraBAD1124::FlgM-6His-TEV-δ-SVIE Δ_(flhDC)8089::(tetR-P_(tetA)) TH18649ΔaraBAD1156::FlgM⁺ Δ_(flhDC)8089::(tetR-P_(tetA)) TH18704ΔaraBAD1156::FlgM⁺ ΔinvH-sprB::FKF(ΔSpi-1) TH18729 ΔaraBAD1156::FlgM⁺ΔfliC7715::tetRA TH18730 ΔaraBAD1156::FlgM⁺ ΔompT::Km TH18731ΔaraBAD1156::FlgM⁺ ΔclpA74::FKF TH18732 ΔaraBAD1156::FlgM⁺ΔsseA-ssaU::FKF(ΔSpi-2) TH18733 ΔaraBAD1156::FlgM⁺ ΔclpP::mini-Tn5TH18737 ΔaraBAD1156::FlgM⁺ ΔfliA5999(R91C, L207P) TH18739ΔaraBAD1156::FlgM⁺ fliA5225(H14D) TH18752 ΔaraBAD1156::FlgM⁺ΔydiV251::tetRA TH18769 ΔaraBAD1124::FlgM-6His-TEV-δ-SVIE ΔfliT::KmTH18778 ΔaraBAD1156::FlgM⁺ fliA5228(V33E) TH18780 ΔaraBAD1156::FlgM⁺fliA5223(T138I) TH18782 ΔaraBAD1156::FlgM⁺ fliA5224(E203D) TH18787ΔaraBAD1156::FlgM⁺ ΔFlgM 5628::FKF TH18788 ΔaraBAD1156::FlgM⁺ ΔfliT::FKFTH18790 ΔaraBAD1156::FlgM⁺ ΔflgK7665 TH18793 ΔaraBAD1156::FlgM⁺ΔflgL7666 TH18796 ΔaraBAD1156::FlgM⁺ ΔfliD5630::FRT TH18798ΔaraBAD1156::FlgM⁺ ΔdegP::tetRA TH18822 ΔaraBAD1156::FlgM⁺ ΔcsrA101::CmTH18823 ΔaraBAD1156::FlgM⁺ ΔdksA::FKF TH18824 ΔaraBAD1156::FlgM⁺ΔrcsB::tetRA TH18830 ΔaraBAD1124::FlgM-6His-TEV-δ-SVIE ΔrcsB::tetRATH18850 ΔaraBAD1156::FlgM⁺ ΔlrhA TH18880ΔaraBAD1124::FlgM-6His-TEV-δ-SVIE P_(flhDC)7793 TH18881ΔaraBAD1156::FlgM⁺ P_(flhDC)7793 TH18897 ΔaraBAD1156::FlgM⁺fliA5240(L199R) TH18973 ΔaraBAD1156::FlgM⁺ ΔecnR5::FCF TH19086ΔaraBAD1156::FlgM⁺ ΔflgN5626::FKF TH19087 ΔaraBAD1156::FlgM⁺ΔfliST5775::FCF TH19099 ΔaraBAD1156::FlgM⁺ Δhin-fljA7731::tetRA TH19104ΔaraBAD1156::FlgM⁺ ΔflgN5626::FKF ΔfliST5775::FCF TH19106ΔaraBAD1156::FlgM⁺ ΔfliS5728::FRT TH19113 ΔaraBAD1156::FlgM⁺Δhin-fljA7731::tetRA ΔfliC7861::FCF TH19116ΔaraBAD1164::FlgM-6His-ETK-δ-SVIE TH19118ΔaraBAD1164::FlgM-6His-ETK-δ-SVIE PflhDC7793 TH19120ΔaraBAD1164::FlgM-6His-ETK-δ-SVIE ΔlrhA ΔecnR4::FRT ΔfliB-T7771P_(flhDC)7793 ΔydiV252 Δhin-fljA7752 ΔFlgM N7753 ΔflgKL7770 TH19145ΔaraBAD1164::FlgM-6His-ETK-δ-SVIE P_(flhDC)7793 ΔFlgM 5628::FKFΔclpX80::tetRA TH19149 ΔaraBAD1156::FlgM⁺ ΔflgKL5636::FKF TH19320ΔaraBAD1156::FlgM⁺ fliA5226(H14N) TH19323 ΔaraBAD1156::FlgM⁺ΔclpA74::FKF ΔflhDC8040::tetRA TH19324 ΔaraBAD1156::FlgM⁺ΔclpP::mini-Tn5 ΔflhDC8040::tetRA TH19325 ΔaraBAD1156::FlgM⁺ΔdegP::tetRA ΔflhDC::FKF TH19326 ΔaraBAD1156::FlgM⁺ Δhin-fljA7731::tetRAΔfliC7861::FCF ΔflgKL5739::FKF TH19481 ΔaraBAD1156::FlgM⁺ ΔfliS8156TH19673 ΔaraBAD1124::FlgM-6His-TEV-δ-SVIE ΔrcsB::tetRA ΔfliT::Km TH19675ΔaraBAD1124::FlgM-6His-TEV-δ-SVIE ΔrcsB::tetRA P_(flhDC)7793 TH20042ΔaraBAD1156::FlgM⁺ Δhin-5717::FRT TH20043 ΔaraBAD1156::FlgM⁺Δhin-5717::FRT ΔfliC7715::tetRA TH20044ΔaraBAD1124::FlgM-6His-TEV-δ-SVIE P_(flhDC)7793 ΔrcsB::tetRA ΔFlgM5628::FKF TH20047 ΔaraBAD1156::FlgM⁺ fljB^(enx) vh2 TH20048ΔaraBAD1124::FlgM-6His-TEV-δ-SVIE fljB^(enx) vh2 TH20049ΔaraBAD1156::FlgM⁺ fljB^(enx) vh2 ΔfliC7715::tetRA TH20050ΔaraBAD1124::FlgM-6His-TEV-δ-SVIE fljB^(enx) vh2 ΔfliC7715::tetRATH20053 ΔaraBAD1156::FlgM⁺ fljB^(enx) vh2 P_(flhDC)7768::tetRA TH20055ΔaraBAD1156::FlgM⁺ fliA8176 (−18 to +1G replaced by ataaAGGAGGtaaataA)TH20056 ΔaraBAD1124::FLgM-6His-TEV-δ-SVIE fliA8176 TH20057ΔaraBAD1156::FlgM⁺ Δhin-5718::FRT TH20058 ΔaraBAD1156::FlgM⁺ fljB^(enx)vh2 ΔPflhDC8089::(tetR-P_(tetA)) TH20059 ΔaraBAD1156::FlgM⁺Δhin-5718::FRT ΔfliC7715::tetRA TH20060 ΔaraBAD1156::FlgM⁺ΔfliC7861::FCF fljB^(enx) vh2 ΔP_(flhDC)8089::(tetR-P_(tetA)) TH20061ΔaraBAD1156::FlgM⁺ fliA8177(H14N, R91C, L207P) TH20062ΔaraBAD1156::FlgM⁺ fliA8178(GTG: ATG, H14N) TH20063 ΔaraBAD1156::FlgM⁺fljB^(enx) vh2 ΔP_(flhDC)8089::(tetR-P_(tetA)) ΔclpX::Tn10dCm TH20064ΔaraBAD1156::FlgM⁺ fljB^(enx) vh2 ΔclpX::Tn10dCm TH20065ΔaraBAD1156::FlgM⁺ fljB^(enx) vh2 ΔydiV251::tetRA TH20066ΔaraBAD1156::FlgM⁺ fljB^(enx) vh2 ΔfliA5805::tetRA TH20067ΔaraBAD1156::FlgM⁺ fljB^(enx) vh2 ΔfliB-T7727::tetRA TH20068ΔaraBAD1156::FlgM⁺ fljB^(enx) vh2 ΔfliC7715::tetRA ΔclpX::Tn10dCmTH20069 ΔaraBAD1156::FlgM⁺ fliA8179(GTG: ATG) TH20071 ΔaraBAD1156::FlgM⁺fljB^(enx) vh2 fliA8176 ΔfliC7861::FCF TH20072 ΔaraBAD1156::FlgM⁺fljB^(enx) vh2 ΔP_(flhDC)8089::(tetR-P_(tetA)) ΔfliA5805::tetRA TH20073ΔaraBAD1156::FlgM⁺ fljB^(enx) vh2 fliA8176 ΔfliC7861::FCF ΔclpX72::FKFTH20074 ΔaraBAD1156::FlgM⁺ ΔFlgM N7753 ΔflgKL7770 Δtrg-7774 ΔycgR7775ΔfliB-T7771 Δhin-fljA7752 fliA8176 TH20075ΔaraBAD1124::FlgM-6His-TEV-δ-SVIE fljB^(enx) vh2 ΔfliB-T7727::tetRATH20077 ΔaraBAD1124::FlgM-6His-TEV-δ-SVIE fljB^(enx) vh2 ΔclpX80::tetRATH20078 ΔaraBAD1156::FlgM⁺ fliA8176 ΔFlgM 5794::FCF TH20079ΔaraBAD1156::FlgM⁺ fljB^(enx) vh2 ΔP_(flhDC)8089::(tetR-P_(tetA))ΔfliA5805::tetRA motA5461::MudJ TH20080 ΔaraBAD1156::FlgM⁺ fljB^(enx)vh2 ΔP_(flhDC)8089::(tetR-P_(tetA)) motA5461::MudJ fliA8176ΔfliC7861::FCF TH20081 ΔaraBAD1156::FlgM⁺ fliA5226H14N ΔfliC7861::FCFTH20082 ΔaraBAD1156::FlgM⁺ fljB^(enx) vh2ΔP_(flhDC)8089::(tetR-P_(tetA)) motA5461::MudJ fliA5226 ΔfliC7861::FCFTH20083 ΔaraBAD1124::FlgM-6His-TEV-δ-SVIE fljB^(enx) vh2ΔP_(flhDC)8089::(tetR-P_(tetA)) ΔclpX::Tn10dCm

ii. Western Blotting Assays.

Levels of secreted, soluble, insoluble or whole cell proteins wereanalyzed by Western blot. Expressed DnaK, FlgM and σ²⁸ levels in thewhole-cell lysates and cultural supernatants were determined by SDS-PAGEusing 14% gradient gels (BIO-RAD). Analysis of strains containingΔaraBAD::FlgM⁺ construct, equivalents of 50 and 100 OD units were loadedfor the cellular and supernatant fractions, respectively. In order toanalyze strains for FlgM-6His-TEV-δ-SVIE and FlgM-6H-ETK-δ-SVIEsecretion, 50 and 300 OD units were loaded for the cellular andsupernatant fractions, respectively. Anti-DnaK (mouse), anti-σ²⁸ andanti-FlgM antibodies (rabbit) were used for detection. DnaK was used asa protein standard control for loading concentration and for thepresence of protein in the supernatant due to cell lysis. To visualizeantigen-antibody complexes, secondary anti-rabbit-IRDye690 andanti-mouse-IRDye800 antibodies (LI-COR) were used. Densitometricmeasurements of FlgM, σ²⁸ and DnaK bands were performed using the LI-COROdyssey Infrared Imaging System software. All assays were performed intriplicate on culture samples.

iii. Motility Assay.

Motility assays utilized soft agar tryptone plates (per liter: 10 gBacto tryptone, 5 g NaCl, 3 g Bacto agar). A bacterial colony was pickedby toothpick and poked through the soft agar followed by incubation at37° C. for about 5 hours. If necessary either arabinose (0.2%) oranhydro-tetracycline (1 μg/ml) was added for P_(araBAD) or flhDC operoninduction, respectively.

2. Results

i. FlgM Produced from P_(araBAD)::FlgM is Secreted.

FlgM is secreted through a completed HBB into the external spent growthmedium. It has been shown that fusion of foreign proteins to theC-terminus of FlgM can be used for protein purification purposes withoutthe need to lyse cells prior to purification. In order to develop theflagellar T3S system for protein purification using FlgM as a secretionsignal, the known aspects of FlgM production and secretion werecharacterized to maximize protein production using this system. The FlgMgene is transcribed from a class 2 flagellar promoter in the flgAMNoperon. This results in FlgM production during initial HBB assembly.Class 2 produced FlgM binds σ²⁸ protein, the product of the fliA gene,which is also produced via class 2 transcription in one of two fliAZtranscripts. Upon HBB completion a change in the secretion substratespecificity of the flagellar T3 S system results in FlgM secretion andinitiation of σ²⁸-dependent class 3 transcription. FlgM and σ²⁸ continueto be produced from σ²⁸-dependent FlgM N and fliAZ transcripts,respectively. About 80% of steady-state FlgM transcription is from itsclass 3 promoter. Since FlgM is an anti-σ²⁸ factor, its production viathe class 3 promoter is under auto-inhibition. In this example, FlgMgene transcription was removed from FlgM auto-inhibition by using aconstruct with the FlgM gene transcribed from the arabinose-induciblechromosomal araBAD promoter (P_(araBAD)). This was accomplished by atargeted deletion of the chromosomal araBAD operon followed by insertionof the FlgM⁺ gene in its place. This resulted in the arabinose-dependentinduction of FlgM production in strains where the arabinose inducer isnot degraded due to deletion of the araBAD structural genes. FlgMtranscribed from P_(araBAD) was produced in the presence of arabinoseand secreted at levels higher than FlgM produced and secreted from itsnative promoters (FIG. 4). A fliF deletion strain, which does not formflagellar structures, was unable to secrete P_(araBAD)-induced FlgM(FIG. 4). These results indicate that FlgM intrinsic-peptide secretionsignals are sufficient for high level FlgM secretion and theflagellar-dependent T3 S system can be used for FlgM secretion.

ii. Mutations Affecting FlhD4C2 Activity Also Affect FlgM Secretion.

The flhDC operon is at the top of the flagellar transcriptionalhierarchy. The regulation of flhDC is complex and there are six knowntranscription initiation sites within the flhDC promoter region. Changesin the −10 sequences for the P1 and P4 transcription initiation siteswithin the flhDC promoter region to the canonical TATAAT sequence (theP_(flhDC)7793 allele) resulted in the doubling of the number of HBBstructures per cell and increased production and secretion of theflagellar hook protein. Other mutations resulting in increased hookprotein secretion and increased HBB structures per cell resulted inreduced expression or removal of known inhibitors of flhDC operontranscriptional or post-transcriptional control. The effects of flhDCregulatory mutations on the secretion of FlgM transcribed fromP_(araBAD), were tested. The known transcriptional andpost-transcriptional inhibitors of flhDC expression included in thisexample were EcnR, RscB, LrhA, FliT, DskA and YdiV. EcnR is responsiblefor FlhDC-mediated auto-repression. The FlhD4C2 complex directstranscription of ecnR, which in turn results in repression of flhDCtranscription in concert with the RcsB protein. RcsB, which regulatescapsular polysaccharide synthesis and a number of genes in response tomembrane and cell wall damage, is a repressor of flhDC transcription.LrhA is also a regulator of flhDC that has been shown to bind within theflhDC promoter region to inhibit flhDC operon transcription. FliT istranscribed from both class 2 and class 3 flagellar promoters. FliTbinds to the FlhD4C2 complex and prevents activation of flagellar class2 transcription. FliT is also the secretion chaperone for the flagellarfilament capping protein FliD. Secretion of FliD after HBB completioncouples inhibition of further class 2 transcription by FlhD4C2 to HBBcompletion. The DskA protein acts with ppGpp to inhibit flhDC'stranscription. DskA can also stimulate rpoS translation, which caninhibit flhDC transcription through an RpoS-mediated mechanism. YdiV isa post-transcriptional regulator that targets FlhD4C2 to the ClpXPprotease for degradation in response to changes in nutrient growthconditions. As a control, CsrA, which is a positive regulator of flhDCmRNA stability, was used. The P_(flhDC)8089 allele was constructed byreplacing the flhDC promoter control region with a tetR-P_(A) cassettefrom transposon Tn10. This resulted in the placement of the flhDC operonunder control of the tetA promoter, which can be induced by the additionof the tetracycline analog anhydro-tetracycline. The flhDC P1 and P4canonical promoter up changes that increase hook production andsecretion (the P_(flhDC)7793 allele) were also tested. The effects ofthe flhDC regulatory mutations on levels of secreted FlgM proteinexpressed from P_(araBAD) are shown in FIG. 5.

Deletion of the flhDC structural operon or deletion of the csrA gene,which is required for flhDC mRNA stability, had no detectable FlgM inthe secreted fraction and FlgM accumulated in the cytoplasm. Mutationsdefective in known transcriptional and post-transcriptional inhibitorsof flhDC expression resulted in increased levels of secreted FlgM as didthe presence of P_(flhDC)7793 promoter-up allele. Secreted levels ofFlgM in P_(flhDC)7793, P_(flhDC)80899 (tetR-P_(tctA)-flhDC), fliT, rcsBand ecnR mutant strains were 4.5-, 4.5-, 3.8-, 4.7- and 4-fold higherthan that of wild type, respectively. Secreted levels of FlgM were lessin ydiV, lrhA and dskA mutants strains at 2-, 1.5- and 2.7-fold comparedto wild type, respectively (FIG. 5B). For each strain tested, theaccumulated cellular levels of FlgM were inversely proportional tosecreted FlgM levels. Significantly, replacement of the flhDC promoterregion with tetA promoter and regulatory region from transposon Tn10allowed for the production of secreted FlgM at levels meeting orexceeding FlgM secreted levels observed in loss-of-function mutants forthe negative regulators of flhDC expression that were tested. Inductionof flhDC in the P_(flhDC)8089 (tetR-P_(tetA)-flhDC) also conferredmotility on swim plates (FIG. 5C).

In strains missing negative regulators of flhDC, the swimming phenotypeson soft agar plates indicated increased motility compared to wild typeLT2 (FIG. 5D). Substantial levels of motility in the same strains withFlgM over-expressed from P_(araBAD) were noted (FIG. 5D). FlgM inhibitsσ²⁸ at a stoichiometry of 1:1. Induction of FlgM from P_(araBAD) canprevent all σ²⁸-dependent flagellar class 3 promoter transcriptionespecially in the wild-type LT2 background. This observation led theconclusion that overexpressed cellular FlgM aggregated into an inactiveform. Thus, the cellular component of FlgM was analyzed from bothsoluble and insoluble fractions from the cell pellet in the Western blotanalysis of secreted and cellular FlgM levels. Although FlgM is asoluble protein most cellular FlgM produced under overexpressionconditions was insoluble (FIG. 5A) indicating that excess cellular FlgMwent into inclusion bodies and the observed motility under FlgM inducingconditions indicated that the insoluble form of FlgM was not active.

iii. The Effect of the FlgM T3S-Chaperone, σ²⁸, on FlgM Secretion.

Completion of the HBB coincides with a flagellar T3Ssubstrate-specificity switch from rod-hook type secretion substrates tolate or filament-type secretion substrates. The late secretionsubstrates include hook-filament junction proteins (FlgK and FlgL),filament cap protein (FliD), the alternately expressed filament proteins(FliC or FljB) and the anti-σ²⁸ factor FlgM. Efficient secretion of eachlate secretion substrates requires the aid of a cognate T3 S chaperone.These include FlgN (for FlgK and FlgL), FliT (for FliD), FliS (for FliCand FljB) and σ²⁸ (for FlgM). T3S chaperones fall into three classes: i)those that bind and protect substrates from proteolysis in the systemprior to secretion, ii) those that facilitate substrate secretion andiii) those that both stabilize and facilitate secretion. The σ²⁸ proteinfalls into the latter category; it protects FlgM from proteolysis in thecytoplasm and facilities the secretion of FlgM presumably by helping tolocalize FlgM to the base of the flagellum. A mutant of σ²⁸ with twoamino acid substitutions that render it defective in recognition of the−10 and −35 promoter sequences (R91C and L207P) retains its T3 Schaperone activity for FlgM secretion. This indicated that the T3Schaperone function of σ²⁸ was a separate process from its transcriptionactivity.

The σ²⁸ protein contains 3 of the 4 regions conserved in all σ⁷⁰-typehousekeeping sigma factors: regions 2, 3 and 4, which are furtherdivided into sub-regions 2.1, 2.2, 2.3, 2.4, 3.1, 3.2, 4.1 and 4.2.Region 2.1, 3.1, 3.2 and 4.1 are involved in binding to the core RNApolymerase while regions 2.4 and 4.2 are required for recognition of the−10 and −35 regions of promoter sequences, respectively. FlgM was shownto interact with all three regions of σ²⁸ in a FlgM:σ²⁸ co-crystalstructure. In addition, single amino acid substitution mutations thatwere defective in FlgM inhibition of σ²⁸-dependent flagellartranscription were located in regions 2.1, 3.1, 4.1 and 4.2. These weredesignated FlgM-bypass mutants because in strains defective for HBBformation they resulted in class 3 flagellar promoter transcription,which is normally inhibited by FlgM in the fliA⁺ background. The σ²⁸FlgMbypass mutants were of two classes. The majority was defective inbinding to FlgM, but two alleles, H14D and H14N each resulted in a σ²⁸protein with increased stability, which was enough to overcome FlgMinhibition.

The effects of FlgM bypass mutants in σ²⁸ regions 2, 3 and 4 onsecretion of FlgM expressed from P_(araBAD) were tested (FIGS. 6A and6B). The region 2.1 mutants included the H14D and H14N, which result inincreased protein stability and the V33E allele, which is defective inbinding FlgM. The other FlgM-bypass mutants tested included the T138Iallele from region 3.1 and L199R from region 4.1 and E203D at theinitiation point of region 4.2.

The fliA null allele showed a strong reduction in FlgM secretion to alevel that was 5% of the fliA⁺ strain. The V33E and L199R alleleresulted in FlgM secretion level that was 12% and 59% of wild type,respectively. The H14D and H14N alleles exhibited secreted FlgM levelsthat were 2.1 and 1.5-fold of that seen for wild type. The T138I andE203D alleles, that are defective in binding FlgM, resulted in increasedsecreted FlgM levels 1.2 and 1.4-fold of wild type, respectively. Thepromoter binding-defective double mutant of fliA (R91C L207P) was alsotested and compared to wild-type showed a 39% level of secreted FlgM.The promoter binding-defective R91C L207P substitutions were alsocombined with the H14D increased stability substitution and observedsecreted FlgM levels in between those observed with the either just theR91C L207P double-mutant or the H14D single mutant.

For wild type FliA, H14N, H14D, R91C L207P, and H14D R91C L207P, theFliA level in the cell was consistent with the FlgM secreted level (FIG.6A). The FlgM secreted level was related to FliA concentration withinthe cell. Therefore, other mutants were introduced, which wouldpotentially increase the intracellular FliA concentration, to seewhether they could affect FlgM secretion. These mutants included a fliAstart codon change from GTG to ATG that was combined with either H14Nstabilization substitution or with a change of the fliA ribosome bindingsequence (RBS) to a canonical sequence (CRBS). All of these changesresulted in increased FliA intracellular levels and secreted FlgM levelsfrom 2- to 4-fold that of wild type (FIGS. 6A and 6B). Deletion of thechromosomal FlgM gene in the fliA CRBS ATG double mutant, backgroundresulted in a reduction of both of intracellular FliA and secreted FlgM.Thus, the excess σ²⁸ produced in the fliA CRBS ATG double mutantbackground can involve chromosomal FlgM expression in addition toP_(araBAD)-expressed FlgM to contribute to σ²⁸ stability, which improvesFlgM secretion. This is consistent with the results showing that FlgMnot only acts as an anti-sigma factor but also protects FliA from beingdegraded.

Motility assays of the fliA mutants under FlgM induced conditions areshowed in FIG. 6C. The fliA null allele and mutants containing the R91CL207P alleles, which are unable to transcribe flagellar class 3promoters and therefore unable to produce flagellin, are non-motileunder any condition. The H14N, H14D, ATG substitution mutants showedincreased swarm sizes on motility plates compared to wild type. V33E,T138I, L199R and E203D are reported to have flagellar transcriptionalactivities in the presence of FlgM that are 8.0, 31, 45 and 7-foldhigher than wild-type, respectively and their motility phenotypes onswim plates under FlgM inducing conditions correlate with these levels.The H14N ATG and CRBS ATG double mutants showed decreased motility onswim plates, although they have higher cellular levels of σ²⁸ than thewild type strain.

iv. The Effect of the Flagellar Late T3S Secretion-Substrate Competitorson FlgM Secretion.

The initial report of FlgM secretion upon HBB completion presentedqualitative data showing that secreted FlgM levels were higher instrains missing filament protein, a potential secretion competitor forFlgM. The effects of removing potential late secretion-substratecompetitors on secreted FlgM levels were tested, to determine if theirremoval would improve the yield of secreted FlgM. The number of latesubstrate subunits in the assembled flagellum are about 11 each forhook-filament junction proteins FlgK and FlgL, 5 for the filamentcapping protein FliD, and, depending on filament length, up to 20,000for FliC or FljB. The results presented in FIGS. 7A and 7B show thatremoval of FlgK, FlgL or FliD had little or no effect on secreted FlgMlevels, while removal of the filament substrates resulted in increasedlevels of secreted FlgM up to 1.9-fold higher than wild-type. Thisresult shows that late T3S substrate levels are not a significantlimiting factor in the secretion of FlgM through the flagella.

The effects of flagellar phase variation on FlgM secretion was alsotested (FIGS. 7A and 7B). Salmonella enterica alternately expresses oneof two flagellin subunit genes, fliC or fljB. Strains carrying thehin-5717 allele are locked in the FliC^(ON) FljB^(OFF) flagellinexpression mode while strains carrying the hin-5718 allele areFliC^(OFF)FljB^(ON). The fljB^(enx)vh2 is a historical relic that isalso FliC^(ON) FljB^(OFF), and resulted from replacement the hin-fljBAregion from S. enterica strain LT2 with the same region from Salmonellaabortus-equi locked in the FliC^(ON)FljB^(OFF) mode. The FlgM secretionlevel in a strain carrying the hin-5717 allele was similar to that ofwild type strain. Deletion of the fliC gene in the hin-5717 backgroundresulted in a 1.9-fold increase in secreted FlgM compared to wild type.The presence of the hin-5718 allele alone increased secreted FlgM levelsby 1.9-fold and the additional removal of the fliC gene in thisbackground further increased the secreted FlgM level 2.7-fold that ofwild type even though FliC flagellin is not produced in the hin-5718background. However, fliC mRNA is produced, but not translated, in thehin-5718 background, showing that fliC mRNA has a negative effect onFlgM protein secretion. Secreted levels of FlgM in the fljB^(enx)vh2background were 2.9-fold higher than in the wild type strain and furtherremoval of the fliC gene in this background increased secreted FlgMlevels to 4.3-fold of wild type.

v. The Effect of the Flagellar Late T3S Chaperones on FlgM Secretion.

The effects of removing the late secretion chaperones FlgN, FliS andFliT on secreted levels of FlgM expressed from P_(araBAD) Was alsotested to determine if they compete with σ²⁸ for delivery of FlgM to theflagellar secretion system for export. Also, T3S chaperones areassociated with regulatory functions in the absence of their cognatesecretion substrates. FlgN, the T3 S chaperone for FlgK and FlgL,inhibits FlgM mRNA translation. σ²⁸ is a transcription factor forflagellar class 3 promoters, and FliT acts as an anti-FlhD4C2 factor.Only FliS is not reported to have a regulatory function in the absenceof its cognate secretion substrates FliC and FljB.

Removal of FlgN had little effect on FlgM secretion (FIGS. 8A and 8B).FlgK and FlgL did not have a significant effect on secreted FlgM levelseither (see FIGS. 7A and 7B). Removal of FliT, which is also reported inFIG. 4, resulted in a 4-fold increase in secreted FlgM levels. Allelesof fliT that separate its anti-FlhD4C2 activity from its chaperoneactivity were not used. Thus, the increase in secreted FlgM levels isdue to enhanced FlhD4C2 activity in the absence of FliT. A 3-foldincrease in secreted FlgM levels in the absence of FliS was observed.However, fliS is transcribed in an operon upstream of the fliT gene.Thus, any polar effect of the fliS alleles on fliT results in increasedFlgM secreted levels due to deceased fliT gene expression.

vi. Deletion of Salmonella Pathogenicity Island 1 (Spi1) Results inIncreased Levels of Secreted FlgM.

The flhDC operon is the master operon of both the flagellar regulon andthe genes of Spi1. The Spi1 regulon encodes genes needed for thestructure and assembly of the Spi1 injectisome T3 S system. The fliZgene is transcribed in the fliAZ operon and FliZ activates transcriptionof hilD, whose product in turn activates Spi1 transcription. One HilDactivated gene product, RtsB, acts to repress flhDC transcriptionproviding a feedback loop for the entire flagellar-Spi1 regulon. Theeffects of deletions of both the Spi1 and Spi2 Salmonella virulencesystems on the secreted levels of overexpressed FlgM (from P_(araBAD))were tested. Loss of Spi1 resulted in decreased FlgM secreted levels to55% that of wild type even though the cells were not grown under Spi1inducing conditions, whereas loss of Spi2 had no significant effect onsecreted levels of FlgM (FIGS. 9A and 9B).

vii. Effect of Protease Removal on FlgM Secreted Levels.

A common technique used to improve protein yield from the cytoplasm isby removing cellular proteases. In addition, proteases present in theouter membrane, such as OmpT can decrease protein yield by degradationafter cell lysis. The ClpA and ClpX proteins interact with differentsubstrates for delivery to the ClpP serine protease for degradation.DegP is a periplasmic protease that exhibits broad substratespecificity. DegP is exclusively directed against unfolded,mis-localized, hybrid and recombinant proteins that are improperlyfolded from over-expression in the periplasm.

The results of protease removal on FlgM secreted levels are shown inFIG. 10. Removal of OmpT resulted in a slight increase the yield ofsecreted FlgM while loss of DegP had little effect. Removal of ClpA,ClpX and ClpP increased the FlgM secretion yield by 1.7-, 5.4- and6.1-fold compared to wild type, respectively (FIGS. 10A and 10B). Thisis consistent with the observations that FlhD4C2 is a substrate forYdiV-directed degradation by the ClpXP protease system. As a control forcell lysis, deletion of flhDC in the protease mutant backgrounds showedno detectable FlgM in the secreted fraction. In this background, thecellular level of FlgM remained unaffected by loss of DegP mutation,while null alleles of clpA, clpX and clpP mutation on the flhDCbackground increased intracellular FlgM accumulation by 1.3-, 1.5- and2.1-fold, respectively (FIG. 10B). The results indicate that ClpA, ClpXand ClpP are involved in FlgM degradation independent of FlhDC; theincreased FlgM secretion in the clpA null strain was due to increasedcellular level FlgM alone. The effects of the ClpXP protease in thepresence of flhD⁺flhC⁻ were due to both FlgM stability and FlhD4C2stability. This is consistent with increased motility observed in theclpP and clpX mutant strains (FIG. 10C).

viii. Effect of Ionic Strength on Secreted FlgM Levels.

High osmolarity increased Spi1 invA gene transcription in S. enterica,and Spi1-dependent type III secretion occurred only in bacteria grownunder high salt conditions. The effects of either increased NaCl or KClon secreted levels of FlgM produced by P_(araBAD)-FlgM⁺ were tested.FlgM secreted levels increased when NaCl concentration was raised to 200mM and then dropped at concentrations of 400 and 600 mM. At 200 mM NaCl,the FlgM secreted level was 3.7-fold higher than the level at 100 mMNaCl (FIGS. 11A and 11B), which is close to the NaCl concentration in astandard LB medium (86 mM). Thus, the NaCl concentration in LB medium isnot optimal for FlgM secretion. KCl had a similar effect on secretedFlgM levels, 200 and 400 mM KCl produced the highest levels of secretedFlgM at 3.2- and 3.6-fold compared to the secreted FlgM level at 100 mMNaCl. This is due to effects on the solubility of FlgM in the cytoplasm.Increased ionic strength also had a positive effect on motility in softagar although this was suppressed under FlgM induction conditions due toinhibition of σ²⁸-dependent flagellar class 3 transcription (FIG. 11C).

ix. Effect of Multiple Conditions on FlgM Secretion.

Individual results described above that improved the yield of secretedFlgM were combined in order to obtain a strain and conditions thatmaximized this yield under FlgM overexpression condition byP_(araBAD)::FlgM⁺ (FIGS. 12A and 12B). Some of these strains, such asfljB^(enx)vh2 and strains containing clpX, strains containing flhD8089,provide the highest FlgM secretion levels. All of them produce secretedFlgM levels about 5-fold that of wild type. In these strains only atrace amount of FlgM accumulated within the cell, showing thatexpression and stability of cellular FlgM was limiting.

x. Use FlgM as a TTS Signal to Secret 8-SVIE.

The δ-SVIE protein is a small peptide produced by a venomous marine conesnail and inhibits sodium channels in vertebrate neuromuscular systems.This 31 amino acid peptide (DGCSSGGTFCGIHPGLCCSEFCFLWCITFID; SEQ IDNO:74) is hydrophobic and contains 6 cysteine residues that form 3 pairsof intramolecular disulfide bonds. The hydrophobic nature of the peptideand requirement for multiple disulfide bond formation to produce anactive conformation of δ-SVIE are an impediment to proper folding whenthis peptide is overexpressed in E. coli. Thus, production of δ-SVIE inan active form via FlgM-mediated secretion was tested. Because secretionfrom the cell initiates with the N-terminus of FlgM, the δ-SVIE sequenceof the FlgM-b-SVIE fusion would exit the cell as it exits the ribosome:from N-terminus to C-terminus This can facilitate proper folding anddisulfide formation of δ-SVIE into an active conformation. C-terminalfusions of δ-SVIE to hexa-His-tagged (6His) FlgM with either TEV or ETKprotease cleavage sites engineered between the two proteins wereexpressed from the chromosomal P_(araBAD) expression locus. The 6Hisfacilitates purification using a Ni-agarose affinity column and the TEVand ETK cleavage sites are recognized by TEV protease and enterokinase,respectively, allowing the secreted δ-SVIE to be separated from its FlgMsecretion signal.

All of the selected single mutants increased FlgM-6H-TEV-δ-SVIEsecretion (see FIGS. 13A and 13B). The secreted levels ofP_(araBAD)-expressed fusion protein levels were enhanced in mutantbackgrounds that produced enhanced secreted FlgM levels. Secreted levelsof FlgM-6H-TEV-δ-SVIE in the flhD7793 (TH18880), flhD8089 (TH18647),fliT (TH18769), rcsB (TH18830), clpX (TH18353) and fliA (CRBS ATG)(TH20056) strains were 16.8-, 10.9-, 9.4-, 6.3-, 16.7-, and 16.6-foldcompared to wild type (TH17831). Different mutations were combinedtogether, to test FlgM-6H-TEV-δ-SVIE secretion levels. In the multiplemutant background strains flhD7793 rcsB (TH19675), flhD7793 rcsB FlgM(TH20044), rcsB FliT (TH19673), fljB^(enx) vh2 fliB-T (TH20075),fljB^(enx) vh2 fliC (TH20050), fljB^(enx) vh2 clpX (TH20077) andflhD8089 fljB^(enx) vh2 clpX (TH20044) were 34-, 17-, 49-, 16.7-, 11.7-,28.2-, and 52.7-fold of that of wild type. When 6His-ETK was insertedbetween FlgM and SVIE, the flhD7793 (TH19118), flhD7793 FlgM (TH19122),flhD7793 FlgM clpX (TH19145), and flhD7793 lrhA fliB-T ydiV hin-fljAFlgM flgKL (TH19120) strains increased FlgM-6H-ETK--SVIE secretionlevels up to 19-, 8.5-, 17-, and 110-fold of that of wild type. Exceptfor the flhD7793 lrhA fliB-T ydiV hin-fljA FlgM N flgKL strain(TH19120), addition of 100 mM NaCl resulted in increased secretionlevels of the fusion protein with strains TH19118 (flhD7793), TH19122(flhD7793 FlgM), and TH19145 (flhD7793 FlgM clpX) were 3.3-, 1.5-, 2.2-and 2.4-fold of wild type.

3. Discussion

The type III secretion (T3S) systems of the flagellum and injectisomeprovide a conduit for the production and purification of recombinantproteins that are fused to T3S signal. There can be a N-terminaldisordered peptide signal and a coupling to the proton motive force asthe secretion fuel. Many, but not all T3S substrates can utilize cognateT3S chaperones for their stability in the cytoplasm and/or as secretionpilots for targeting the T3S substrate to the secretion apparatus in thecytoplasmic membrane. Another feature of T3S systems is the ability toundergo a secretion-specificity switch from early to late secretionsubstrate specificity. In the flagellar system this occurs uponcompletion of extracellular hook growth that is more than 40 nm inlength resulting in the transition to filament-type substrate secretionand assembly. The FlgM protein is an integral part of the transitionfrom hook completion to filament assembly. FlgM is a late, filament-typeflagellar secretion substrate and an anti-σ²⁸ factor. FlgM is a small,97 amino acid protein. The N-terminal half of FlgM includes the T3Ssignal while the C-terminal half includes the anti-σ²⁸ interactiondomain. It has been shown that fusion of recombinant proteins to theC-terminus of FlgM would allow for their secretion and purificationwithout a requirement to lyse cells prior to purification. Conditionsthat facilitate FlgM production and secretion were examined. Theseconditions were then applied to produce the recombination proteinsFlgM-6His-TEV-δ-SVIE and FlgM-6His-ETK-δ-SVIE to produce and purifythese proteins without cell lysis using FlgM as a vector to direct thesecretion of the −δ-SVIE proteins from the cell via the flagellar T3Ssystem.

FlgM and FlgM-δ-SVIE fusion constructs were over-expressed from thechromosomal P_(araBAD) promoter by replacing the araBAD coding regionwith the FlgM or FlgM-δ-SVIE fusion coding regions. By removing araBADit was ensured that arabinose inducer would not be consumed as a carbonsource. This inducing system proved sufficient to produce FlgM inquantities in excess of what the cell could secrete (FIG. 5). The cellswere then manipulated by introducing mutations that increase FlgMstability and secretion in order to increase production of secretedFlgM.

The effect of mutations that increase the number of flagellar T3 Ssystems per cell on secreted FlgM levels were examined. These includednull mutations in negative regulators of flhDC transcription (ecnR,rcsB, lrhA, dskA) and flhDC promoter-up alleles P_(flhDC)7793 andP_(flhDC)8089. Null alleles of fliT and ydiV that inhibit FlhD4C2function at a post-transcriptional level were also tested. Suchmutations had been previously shown to increase the production andsecretion of the flagellar hook protein into the periplasm. All mutantbackgrounds tested resulted in increased levels of secreted FlgM. TheP_(flhDC)8089 allele is a replacement of the flhDC promoter region withan inducible tetA promoter region, which removes the site of action ofall known negative regulators of flhDC transcription. The P_(flhDC)8089allele also places flhDC transcription under induction by tetracyclineand its analogs. Without the inducer, the cells were not motile onswimming plate, indicating that flhDC was not transcribed. FlgM was notsecreted in the csrA null mutant strain, as CsrA can stabilize flhDCmRNA.

The effects of various alleles of the FlgM T3S chaperone σ²⁸ encoded bythe fliA gene were explored. These included FlgM bypass alleles of fliAthat allow class 3 transcription in the presence of FlgM, the promoterbinding-defective double-mutant R91C L207P, the ATG start codon mutant(from GTG), and a canonical ribosome binding sequence mutant (labeledCRBS). The FlgM bypass alleles H14D, H14N, V33E, T138I, L199R and E203Dall have similar affinities for RNAP with a measured K_(d) for core RNAPof 2.0, 1.9, 0.7, 0.8, 1.2 and 1.4 nM, respectively compared to 0.9 forwild-type σ²⁸. The relative affinities for FlgM of the H14D and H14N arethe same as wild-type, while the V33E, T138I, L199R and E203D alleleshave 10-, 20-, 600- and 10-fold reduced affinities, respectively. TheV33E allele showed the lowest levels of secreted FlgM while L199R hadabout half the secreted level of FlgM as wild-type. However, the lowcellular level of V33E σ²⁸ allele shows that it is a limiting σ²⁸ thatis responsible for the reduced secreted FlgM levels observed. Thereduced affinity of T138I and V203E for FlgM is not a limiting factorunder FlgM over-expressing conditions (from P_(araBAD)). The R91C L207Ppromoter binding-defective double-mutation resulted in reduced σ²⁸cellular levels that corresponded to a reduced level of secreted FlgMwhile the H14D and H14N alleles had the opposite effect. The H14D andH14N alleles resulted in elevated cellular σ²⁸ levels and correspondedto increased levels of secreted FlgM. Addition of H14D or H14N to theR91C L207P double-mutant resulted in increased σ²⁸ levels and secretedlevels of FlgM. The ATG with or without the additional H14N substitutionor the canonical ribosome binding sequence mutant (ATG CRBS) allresulted in elevated cellular σ²⁸ and secreted FlgM levels. Theseresults show that increasing cellular levels of σ²⁸ results in acorresponding increase in FlgM secretion under FlgM over-expressionconditions.

Removal of late secretion competitors of FlgM secretion or their cognatechaperones had mixed results. Of the 4 secretion competitors FlgK, FlgL,FliD and FliC/FljB only removal of the filament late secretionsubstrates FliC/FljB had a significant effect of FlgM secretion. Theamount of filament subunits in the flagellum is about 1000 times that ofthe other three components. Removal of the FliD secretion chaperone FliTalso increased FlgM secretion independent of its role on increasing thestability of FliD. FliT is a regulator of FlhD4C2 promoter activationand its removal results in increased HBB secretion conduits per cell.Removal of filament T3S chaperone protein, FliS, resulted in a 3-foldincrease in secreted FlgM levels while removal of the FlgK and FlgL T3 Schaperone had no effect. As fliS and fliT are cotranscribed in the sameoperon (fliS is upstream of fliT), any deletion mutation of fliS mayaffect fliT gene expression. A flagellar phase variation mutant allele,fljB^(enx)2, showed a significant increase in secreted FlgM levelscompared to the hin-5717 allele. Increased secreted FlgM levels werealso observed in the Spi1, even when the cells were grown under Spi1non-inducing conditions.

Removal of cellular proteases also produced mixed results on FlgMsecretion. The ClpXP protease regulates the number of flagella per cellby degradation of the FlhD4C2 complex, which is directed by the YdiVprotein. YdiV is produced during poor nutrient growth conditions. YdiVbinds the FlhD component of the FlhD4C2, which prevents furtherinteraction between FlhD4C2 and DNA. YdiV then targets FlhD4C2 to ClpXPprotease for degradation. Removal of either ClpX or ClpP resultedincreased FlgM secreted levels. Removal of DegP or OmpT proteases werealso tested for effects on FlgM secretion and no effect was observed.

The last variable tested on secreted levels of over-expressed FlgM wasionic strength. Type III secretion was induced by high osmolarity. Theeffect of NaCl and KCl concentration on FlgM secretion was tested and itwas observed that addition of NaCl to 200 mM or KCl to 200-400 mMresulted in a about 4-fold increase in secreted FlgM levels compared to100 mM NaCl, which is close to the concentration of NaCl in LB (0.5% or86 mM). The NaCl effect was due to increased potential of the protonmotive force. However, the same effect was observed with the addition ofKCl showing that it is simply ionic strength that controls FlgM secretedlevels. Ionic strength can result in increased stability of FlhD4C2.

After determining the effects of different mutations on secreted levelsof FlgM the observations were combined to construct some strains thatmaximize the amount of FlgM secreted from the cell. All of the strainscontaining fliB-T, clpX, or flhD8089 increased the FlgM secretion about5-fold of that of wild type strain, and the combination did not increaseFlgM secretion further more. This is because nearly all of the FlgM wassecreted, and only trace amount of FlgM accumulated in the cell. So whendifferent mutations were combined together, the expression level of theFlgM became limiting factor of FlgM secretion level.

Finally, a disulfide-rich small peptide 6-SVIE contoxin was fused toFlgM, to test whether the peptide can be secreted and how the mutationsand ion concentration affect its secretion. Both FlgM-6His-TEV-δ-SVIEand FlgM-6His-ETK-δ-SVIE can be secreted to the medium, and themutations which stimulated FlgM⁺ secretion also increased these fusionproteins secretion. Some combined mutants such as rcsB fliT strain andflhD8089 fljB^(enx) vh2 clpX strain increased FlgM-6H-TEV-δ-SVIEsecretion about 50-fold compare to wild type. Another combined strainflhD7793 lrhA ecnR fliB-T ydiV hin-fljA flgKL FlgM N increased thesecretion of FlgM-6H-ETK-δ-SVIE over 100-fold compare to wild typestrain. The addition of 100 mM NaCl to LB medium can increase thesecretion of FlgM-6H-ETK-δ-SVIE in flhD7793 strain, flhD7793 FlgMstrain, and flhD7793 FlgM clpX strain. These results indicated that FlgMcan be used as a T3S signal to express and purify proteins which aredifficult or impossible to do via E coil overexpression system.

C. Example 3 Large-Scale, Type III-Dependent Protein Production ViaDirect Secretion into the Growth Medium

The use of FlgM as a secretion signal to facilitate secretion ofpreviously difficult to produce proteins, such as conopeptides withnumerous disulfide bonds and the A subunit of diphtheria toxin, which isnot detectable in the cytoplasm due to its instability, demonstrates theutility of this system for protein production and purification ofproteins that otherwise are not produced.

Using lambda RED technology for chromosomal targeting, this system canrevolutionize bacterial genetic strain construction. In the desiredgenetic background the araBAD operon can be replaced by a tetRA element.The tetRA element can be the tetR and tetA genes from transposon Tn10that code for a tetracycline efflux pump (TetA), which confersresistance to tetracycline, and the TetR repressor. This can beaccomplished by PCR-amplification of the tetRA element using 48-meroligonucleotides. One ologo has 40 bases of homology to the 5′ side ofthe beginning of the araB gene followed by 18 bases that can amplifyfrom the 5′-tetR end of tetRA. The second has 40 bases of homology tothe 3′ side of the end of the araD gene followed by 18 bases that canamplify from the 3′-tetA end of tetRA. PCR amplification oftetRA-containing DNA produces the tetRA element flanked by 40 bases ofhomology to the target. Introduction of the amplified fragment into astrain that expresses the lambda recombination genes (RED) results inrecombination using the 40 bases of flanking homology resulting indeletion of the araBAD genes that are replaced by tetRA (ΔaraBAD::tetRAwhere A is genetic nomenclature for deletion and :: is geneticnomenclature for insertion). The advantage of the tetRA element is thatit can be selected for and against. The presence of the tetRA element inthe chromosome confers tetracycline resistance, but also conferssensitivity of fusaric acid. Thus, when the FlgM coding sequence is PCRamplified with the same 40 bases of homology 5′ to araB and 3′ to araDin the presence of lambda RED and plated on fusaric acid medium, thisselects for recombination that replaces the tetRA of ΔaraBAD::tetRA withFlgM⁺ sequence resulting in ΔaraBAD::FlgM where the FlgM⁺ gene is nowtranscribed from the arabinose-inducible araBAD promoter. Next, by thesame method tetRA is placed downstream of FlgM⁺ at araBAD resulting inΔaraBAD::FlgM⁺-tetRA followed by replacement that tetRA with protein Xresulting in a FlgM-X fusion that is expressed by addition of arabinoseto the growth medium. Using this system any fusion of any protein toFlgM under arabinose induction can be constructed with interveningprotease cleavage sequences to facilitate final purification of aprotein of interest.

1. Strain & Conditions Optimizations.

Optimum secretion strains can be identified. The promoter up allele ofthe flhDC promoter, flhD7793, and the allele resulting from thereplacement of the flhDC promoter with the anhydrotetracycline (AnTc)inducible tetA promoter, flhD8089, each results in substantial increasein FlgM secretion that is greater than that observed by removal ofinhibitors of flhDC transcription: FliT, LrhA, DskA, RcsB or EcnR (FIG.5A). The AnTc-inducible flhDC operon (the flhD8089 allele) is of notebecause it is deleted for the binding sites of all known inhibitorproteins and dependent only on AnTc for expression. The flhD8089 allelecan be combined with the fliA(α²⁸) H14N allele of the secretionchaperone of FlgM, which showed to greatest positive effect on FlgMsecretion (FIG. 6A). Further combination with a clpX protease mutantallele can increase secretion (FIG. 10A). Using this genetic backgroundin a strain that also expresses a protein of interest fused to the FlgMflagellar type Ill secretion signal can increase production of theprotein of interest, especially if grown in the presence of 200 mM NaClor KCl (FIG. 11A).

2. The σ²⁸ Purification Column.

The σ²⁸ protein is the product of the fliA gene and the type IIIsecretion chaperone for FlgM. Increased σ²⁸ levels result in increasedlevels of secreted FlgM-fused protein substrate (FIG. 6A). The σ²⁸protein can also be used for a protein purification system. The apparentK_(d) of the FlgM-σ²⁸ complex is 2×10⁻¹⁰ M. This remarkably highaffinity means that purification of FlgM can result in co-purificationof σ²⁸ and vice versa. FlgM fused to a chitin-binding domain (CBD)sandwiched by an intein self-cleavage site has been constructed. Whenextracts from cells expressing this fusion chimera were poured over achitin column the FlgM-intein-CBD chimera in complex with σ²⁸ bound thecolumn. Addition of a reducing agent catalyzed intein self-cleavagereleasing purified FlgM-σ²⁸ complex. Making a chitin column bound byσ²⁸-CBD can use this aspect. The spent growth medium of FlgM-intein-Xchimera produced from our flagellar secretion strain, where X is theprotein of interest, can be poured over such a column. Induction ofintein self-cleavage can release protein X in pure form. This canprovide a low-cost high-yield protein production system.

Proteins that must remain in oxidative conditions to maintain an activeconformation can have a protease cleavage sequence, such asenterokinase, which cleaves under oxidative conditions, in place of theintein.

The disclosed system and methods can also be developed into E. coli toobtain the protein secretion and production with the E. coli flagellarT3 S system that was achieved in Salmonella. This can be expanded toinclude other strains such as thermo-tolerant strains. Proteins fromthermo tolerant strains are often used in crystallographic and NMRstudies because of increased protein stabilities at high temperaturesthat facilitate structural analysis.

A SPI1 injectisome T3S system for protein production can be developed.The SipA, SipB and SipC proteins are secreted at high levels by the SPI1injectisome T3S system (FIG. 14). These proteins can be tested todetermine if they can be used to facilitate protein secretion in fusionconstructs and develop a protein production system similar to theflagellar system. Spi1 production is under the control of HilD and ithas been determined that controlled expression of hilD⁺ from thearabinose-inducible P_(araBAD) promoter results in high levels of Spi1mRNA production.

E. coli has essentially the identical flagellar T3S as Salmonella,making it straightforward to reconstruct the optimal secretion strain inE. coli. However, E. coli lacks the SPI1 system and thus the entire SPI1set of genes can be inserted into the E. coli chromosome. As shown inFIG. 15A, the SPI1 genes are organized into a single “pathogenicityisland” which can be amplified and inserted into the E. coli chromosomeusing the lambda RED technology. The export apparatus and needle complexgenes can be used, while the hilA+ gene will be inserted into the araBADlocus to allow for arabinose induction of SPI1.

The SPI1 system can also be used to develop a membrane-proteinpurification system. Such as system can revolutionize membrane proteinproduction and characterization. The SPI1 system works by inserting intothe cytoplasmic membrane of host cells through its translocon tip (seeFIG. 16). Modifying injectisome needle-length can be done in order todevelop a minimal functional needle that is as short as possible yetstill allows for normal translocon formation and insertion into themembrane of host cells. Minimizing the needle length through which amembrane protein must traverse can be beneficial. This can be coupled totranslocation of secretion of membrane proteins using the SPI1 systeminto artificial membrane vesicles.

REFERENCES

-   1. Abramoff, M. D., P. J. Magelhaes, and S. J. Ram. 2004. Image    Processing with ImageJ. Biophotonics International 11:36-42.-   2. Aldridge, P. D., J. E. Karlinsey, C. Aldridge, C. Birchall, D.    Thompson, J. Yagasaki, and K. T. Hughes. 2006. The    flagellar-specific transcription factor, sigma28, is the Type III    secretion chaperone for the flagellar-specific anti-sigma28 factor    FlgM. Genes Dev. 20:2315-2326.-   3. Bulaj, G., P. J. West, J. E. Garrett, M. Watkins, M. Marsh, M.-M.    Zhang, R. S. Norton, B. J. Smith, D. Yoshikami, and B. M.    Olivera. 2005. Novel conotoxins from Conus striatus and Conus    kinoshitai selectively block TTX-resistant sodium channels.    Biochemistry 44:7259-7265.-   4. Chadsey, M. S., and K. T. Hughes. 2001. A multipartite    interaction between Salmonella transcription factor sigma28 and its    anti-sigma factor FlgM: implications for sigma28 holoenzyme    destabilization through stepwise binding. Journal of Molecular    Biology 306:915-929.-   5. Chadsey, M. S., J. E. Karlinsey, and K. T. Hughes. 1998. The    flagellar anti-sigma factor FlgM actively dissociates Salmonella    typhimurium sigma28 RNA polymerase holoenzyme. Genes Dev    12:3123-3136.-   6. Chahine, M., L. Q. Chen, N. Fotouhi, R. Walsky, D. Fry, V.    Santarelli, R. Horn, and R. G. Kallen. 1995. Characterizing the    mu-conotoxin binding site on voltage-sensitive sodium channels with    toxin analogs and channel mutations. Recept Channels 3:161-174.-   7. Chahine, M., J. Sirois, P. Marcotte, L. Chen, and R. G.    Kallen. 1998. Extrapore residues of the S5-S6 loop of domain 2 of    the voltage-gated skeletal muscle sodium channel (rSkM1) contribute    to the mu-conotoxin GIIIA binding site. Biophys J 75:236-246.-   8. Chang, N. S., R. J. French, G. M. Lipkind, H. A. Fozzard, and S.    Dudley, Jr. 1998. Predominant interactions between mu-conotoxin    Arg-13 and the skeletal muscle Na+ channel localized by mutant cycle    analysis. Biochemistry 37:4407-4419.-   9. Che, N., L. Wang, Y. Gao, and C. An. 2009. Soluble expression and    one-step purification of a neurotoxin Huwentoxin-I in Escherichia    coli. Protein Expression and Purification 65:154-159.-   10. Chevance, F. F. V., and K. T. Hughes. 2008. Coordinating    assembly of a bacterial macromolecular machine. Nat Rev Microbiol    6:455-465.-   11. Dobo, J., J. Varga, R. Sajo, B. M. Vegh, P. Gal, P. Zavodszky,    and F. Vonderviszt. 2010. Application of a short, disordered    N-terminal flagellin segment, a fully functional flagellar type III    export signal, to expression of secreted proteins. Applied and    Environmental Microbiology 76:891-899.-   12. Dudley, S. C., H. Todt, G. Lipkind, and H. A. Fozzard. 1995. A    mu-conotoxin-insensitive Na+ channel mutant: possible localization    of a binding site at the outer vestibule. Biophys J 69:1657-1665.-   13. Erhardt, M., and K. T. Hughes. 2010. C-ring requirement in    flagellar type III secretion is bypassed by FlhDC upregulation. Mol    Microbiol 75:376-393.-   14. Erhardt, M., K. Namba, and K. T. Hughes. 2010. Bacterial    nanomachines: the flagellum and type Ill injectisome. Cold Spring    Harb Perspect Biol 2:a000299.-   15. Fiedler, B., M.-M. Zhang, O. Buczek, L. Azam, G. Bulaj, R. S.    Norton, B. M. Olivera, and D. Yoshikami. 2008. Specificity, affinity    and efficacy of iota-conotoxin RXIA, an agonist of voltage-gated    sodium channels Na(V)1.2, 1.6 and 1.7. Biochem Pharmacol    75:2334-2344.-   16. Frye, J., J. E. Karlinsey, H. R. Felise, B. Marzolf, N.    Dowidar, M. McClelland, and K. T. Hughes. 2006. Identification of    new flagellar genes of Salmonella enterica serovar Typhimurium. J    Bacteriol 188:2233-2243.-   17. Green, B. R., P. Catlin, M.-M. Zhang, B. Fiedler, W. Bayudan, A.    Morrison, R. S. Norton, B. J. Smith, D. Yoshikami, B. M. Olivera,    and G. Bulaj. 2007. Conotoxins containing nonnatural backbone    spacers: cladistic-based design, chemical synthesis, and improved    analgesic activity. Chemistry & Biology 14:399-407.-   18. Hughes, K. T., K. L. Gillen, M. J. Semon, and J. E.    Karlinsey. 1993. Sensing structural intermediates in bacterial    flagellar assembly by export of a negative regulator. Science    262:1277-1280.-   19. Hui, K., G. Lipkind, H. A. Fozzard, and R. J. French. 2002.    Electrostatic and steric contributions to block of the skeletal    muscle sodium channel by mu-conotoxin. J Gen Physiol 119:45-54.-   20. Jones, R. M., and G. Bulaj. 2000. Conotoxins—new vistas for    peptide therapeutics. Curr Pharm Des 6:1249-1285.-   21. Karlinsey, J. E. 2007. lambda-Red genetic engineering in    Salmonella enterica serovar Typhimurium. Meth Enzymol 421:199-209.-   22. Karlinsey, J. E., S. Tanaka, V. Bettenworth, S. Yamaguchi, W.    Boos, S. I. Aizawa, and K. T. Hughes. 2000. Completion of the    hook-basal body complex of the Salmonella typhimurium flagellum is    coupled to FlgM secretion and fliC transcription. Mol. Microbiol.    37:1220-1231.-   23. Kutsukake, K. 1994. Excretion of the anti-sigma factor through a    flagellar substructure couples flagellar gene expression with    flagellar assembly in Salmonella typhimurium. Mol Gen Genet    243:605-612.-   24. Lee, H. J., and K. T. Hughes. 2006. Posttranscriptional control    of the Salmonella enterica flagellar hook protein FlgE. J Bacteriol    188:3308-3316.-   25. Lehnen, D., C. Blumer, T. Polen, B. Wackwitz, V. F. Wendisch,    and G. Unden. 2002. LrhA as a new transcriptional key regulator of    flagella, motility and chemotaxis genes in Escherichia coli. Mol    Microbiol 45:521-532.-   26. Miljanich, G. 1997. Venom peptides as human pharmaceuticals. Sci    Med 4:6.-   27. Miljanich, G. P. 2004. Ziconotide: neuronal calcium channel    blocker for treating severe chronic pain. Curr Med Chem    11:3029-3040.-   28. Nakamura, M., Y. Niwa, Y. Ishida, T. Kohno, K. Sato, Y. Oba,    and H. Nakamura. 2001. Modification of Arg-13 of mu-conotoxin GIIIA    with piperidinyl-Arg analogs and their relation to the inhibition of    sodium channels. FEBS Left 503:107-110.-   29. Ohnishi, K., K. Kutsukake, H. Suzuki, and T. Lino. 1992. A novel    transcriptional regulation mechanism in the flagellar regulon of    Salmonella typhimurium: an antisigma factor inhibits the activity of    the flagellum-specific sigma factor, sigma F. Mol Microbiol    6:3149-3157.-   30. Olivera, B. 2000. ω-Conotoxin MVIIA: From Marine Snail Venom to    Analgesic Drug. Drugs from the Sea (Fusetani, N., ed):pp. 77-85.-   31. Sanderson, K. E., and J. R. Roth. 1983. Linkage map of    Salmonella typhimurium, Edition VI. Microbiol. Rev. 47:410-453.-   32. Takaya, A., M. Erhardt, K. Karata, K. Winterberg, T. Yamamoto,    and K. T. Hughes. 2012. YdiV: a dual function protein that targets    FlhDC for ClpXP-dependent degradation by promoting release of    DNA-bound FlhDC complex. Molecular microbiology 83:1268-1284.-   33. Terlau, H., and B. M. Olivera. 2004. Conus venoms: a rich source    of novel ion channel-targeted peptides. Physiol Rev 84:41-68.-   34. Wozniak, C., C. Lee, and K. Hughes. 2008. T-POP array identifies    EcnR and PefI-SrgD as novel regulators of flagellar gene expression.    J Bacteriol.-   35. Yao, S., M.-M. Zhang, D. Yoshikami, L. Azam, B. M. Olivera, G.    Bulaj, and R. S. Norton. 2008. Structure, dynamics, and selectivity    of the sodium channel blocker mu-conotoxin SIIIA. Biochemistry    47:10940-10949.-   36. Frye, J., J. E. Karlinsey, H. R. Felise, B. Marzolf, N.    Dowidar, M. McClelland, and K. T. Hughes. 2006. Identification of    new flagellar genes of Salmonella enterica serovar Typhimurium. J    Bacteriol 188:2233-2243.-   1. Aldridge, C., K. Poonchareon, S. Saini, T. Ewen, A.    Soloyva, C. V. Rao, K. Imada, T. Minamino, and P. D. Aldridge. 2010.    The interaction dynamics of a negative feedback loop regulates    flagellar number in Salmonella enterica serovar Typhimurium. Mol.    Microbiol. 78:1416-1430.-   2. Aldridge, P. D., J. E. Karlinsey, C. Aldridge, C. Birchall, D.    Thompson, J. Yagasaki, and K. T. Hughes. 2006. The    flagellar-specific transcription factor, sigma28, is the Type III    secretion chaperone for the flagellar-specific anti-sigma28 factor    FlgM. Genes Dev. 20:2315-2326.-   3. Auvray, F., J. Thomas, G. M. Fraser, and C. Hughes. 2001.    Flagellin polymerisation control by a cytosolic export chaperone. J.    Mol. Biol. 308:221-229.-   4. Baneyx, F., and G. Georgiuo. 1990. In vivo degradation of    secreted fusion proteins by the Escherichia coli outer membrane    protease OmpT. J. Bacteriol. 172:491-494.-   5. Barembruch, C., and R. Hengge. 2007. Cellular levels and activity    of the flagellar sigma factor FliA of Escherichia coli are    controlled by FlgM-modulated proteolysis. Mol. Microbiol. 65:76-89.-   6. Berg, H. C., and R. A. Anderson. 1973. Bacteria swim by rotating    their flagellar filaments. Nature 245:380-382.-   7. Bonifield, H. R., and K. T. Hughes. 2003. Flagellar phase    variation in Salmonella enterica serovar Typhimurium is mediated by    a posttranscriptional control mechanism. J. Bacteriol.    185:3567-3574.-   8. Chadsey, M. S., and K. T. Hughes. 2001. A multipartite    interaction between Salmonella transcription factor sigma28 and its    anti-sigma factor FlgM: implications for sigma28 holoenzyme    destabilization through stepwise binding. J. Mol. Biol. 306:915-929.-   9. Chevance, F. F., and K. T. Hughes. 2008. Coordinating assembly of    a bacterial macromolecular machine. Nat. Rev. Microbiol. 6:455-465.-   10. Chubiz, J. E., Y. A. Golubeva, D. Lin, L. D. Miller, and J. M.    Slauch. 2010. FliZ regulates expression of the Salmonella    pathogenicity island 1 invasion locus by controlling HilD protein    activity in Salmonella enterica serovar typhimurium. J. Bacteriol.    192:6261-6270.-   11. Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation    of chromosomal genes in Escherichia coli K-12 using PCR products.    Proc. Natl. Acad. Sci. USA 97:6640-6645.-   12. Daughdrill, G. W., M. S. Chadsey, J. E. Karlinsey, K. T. Hughes,    and F. W. Dahlquist. 1997. The C-terminal half of the anti-sigma    factor, FlgM, becomes structured when bound to its target, sigma 28.    Nat. Struct. Biol. 4:285-291.-   13. Davis, R. W., D. Botstein, and J. R. Roth. 1980. Advanced    Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring    Harbor, N.Y.-   14. Dorel, C., P. Lejeune, and A. Rodrigue. 2006. The Cpx system of    Escherichia coli, a strategic signaling pathway for confronting    adverse conditions and for settling biofilm communities?Res.    Microbiol. 157:306-314.-   15. Ellermeier, C. D., and J. M. Slauch. 2003. RtsA and RtsB    coordinately regulate expression of the invasion and flagellar genes    in Salmonella enterica serovar Typhimurium. J. Bacteriol.    185:5096-5108.-   16. Enomoto, M., and B. A. Stocker. 1975. Integration, at hag or    elsewhere, of H2 (phase-2 flagellin) genes transduced from    Salmonella to Escherichia coli. Genetics 81:595-614.-   17. Erhardt, M., and K. T. Hughes. 2010. C-ring requirement in    flagellar type III secretion is bypassed by FlhDC upregulation. Mol.    Microbiol. 75:376-393.-   18. Fattori, J., A. Prando, A. M. Martins, F. H. Rodrigues, and L.    Tasic. 2011. Bacterial secretion chaperones. Protein Pept. Lett.    18:158-166.-   19. Flynn, J. M., S. B. Neher, Y. I. Kim, R. T. Sauer, and T. A.    Baker. 2003. Proteomic discovery of cellular substrates of the ClpXP    protease reveals five classes of ClpX-recognition signals. Mol. Cell    11:671-683.-   20. Francez-Charlot, A., B. Laugel, A. Van Gemert, N. Dubarry, F.    Wiorowski, M. P. Castanie-Cornet, C. Gutierrez, and K. Cam. 2003.    RcsCDB His-Asp phosphorelay system negatively regulates the flhDC    operon in Escherichia coli. Mol. Microbiol. 49:823-832.-   21. Fraser, G. M., J. C. Bennett, and C. Hughes. 1999.    Substrate-specific binding of hook-associated proteins by FlgN and    FliT, putative chaperones for flagellum assembly. Mol. Microbiol.    32:569-580.-   22. Galimn, J. E., and R. r. Curtiss. 1990. Expression of Salmonella    typhimurium genes required for invasion is regulated by changes in    DNA supercoiling. Infect. Immun 58:1879-1885.-   23. Gillen, K. L., and K. T. Hughes. 1991. Molecular    characterization of FlgM, a gene encoding a negative regulator of    flagellin synthesis in Salmonella typhimurium. J. Bacteriol.    173:6453-6459.-   24. Gillen, K. L., and K. T. Hughes. 1993. Transcription from two    promoters and autoregulation contribute to the control of expression    of the Salmonella typhimurium flagellar regulatory gene FlgM. J.    Bacteriol. 175:7006-7015.-   25. Hughes, K. T., A. Dessen, J. P. Gray, and C. Grubmeyer. 1993.    The Salmonella typhimurium nadC gene: sequence determination by use    of Mud-P22 and purification of quinolinate    phosphoribosyltransferase. J. Bacteriol. 175:479-486.-   26. Hughes, K. T., K. L. Gillen, M. J. Semon, and J. E.    Karlinsey. 1993. Sensing structural intermediates in bacterial    flagellar assembly by export of a negative regulator. Science    262:1277-1280.-   27. Ikebe, T., S. Iyoda, and K. Kutsukake. 1999. Structure and    expression of the fliA operon of Salmonella typhimurium. Microbiol.    145:1389-1396.-   28. Iyoda, S., T. Kamidoi, K. Hirose, K. Kutsukake, and H.    Watanabe. 2001. A flagellar gene fliZ regulates the expression of    invasion genes and virulence phenotype in Salmonella enterica    serovar Typhimurium. Microb. Pathog. 30:81-90.-   29. Karlinsey, J. E. 2007. lambda-Red genetic engineering in    Salmonella enterica serovar Typhimurium. Meth. Enzymol. 421:199-209.-   30. Karlinsey, J. E., J. Lonner, K. L. Brown, and K. T. Hughes.    2000.

Translation/secretion coupling by type III secretion systems. Cell102:487-497.

-   31. Kutsukake, K. 1994. Excretion of the anti-sigma factor through a    flagellar substructure couples flagellar gene expression with    flagellar assembly in Salmonella typhimurium. Mol. Gen. Genet.    243:605-612.-   32. Lehnen, D., C. Blumer, T. Polen, B. Wackwitz, V. F. Wendisch,    and G. Unden. 2002. LrhA as a new transcriptional key regulator of    flagella, motility and chemotaxis genes in Escherichia coli. Mol.    Microbiol. 45:521-532.-   33. Lemke, J. J., T. Durfee, and R. L. Gourse. 2009. DksA and ppGpp    directly regulate transcription of the Escherichia coli flagellar    cascade. Mol. Microbiol. 74:1368-1379.-   34. Lucas, R. L., C. P. Lostroh, C. C. DiRusso, M. P. Spector, B. L.    Wanner, and C. A. Lee. 2000. Multiple factors independently regulate    hilA and invasion gene expression in Salmonella enterica serovar    typhimurium. J. Bacteriol. 182:1872-1882.-   35. Macnab, R. M. 2003. How bacteria assemble flagella. Annu. Rev.    Microbiol. 57:77-100.-   36. Macnab, R. M. 2004. Type III flagellar protein export and    flagellar assembly. Biochim. Biophys. Acta 1694:207-217.-   37. Merdanovic, M., T. Clausen, M. Kaiser, R. Huber, and M.    Ehrmann. 2011. Protein quality control in the bacterial periplasm.    Annu. Rev. Microbiol. 65:149-168.-   38. Minamino, T., and K. Namba. 2008. Distinct roles of the ATPase    and proton motive force in bacterial flagellar protein export.    Nature 451:485-488.-   39. Namba, K. 2001. Roles of partly unfolded conformations in    macromolecular self-assembly. Genes Cells 6:1-12.-   40. Ohnishi, K., K. Kutsukake, H. Suzuki, and T. Lino. 1990. Gene    fliA encodes an alternative sigma factor specific for flagellar    operons in Salmonella typhimurium. Mol. Gen. Genet. 221:139-147.-   41. Ohnishi, K., K. Kutsukake, H. Suzuki, and T. Lino. 1992. A novel    transcriptional regulation mechanism in the flagellar regulon of    Salmonella typhimurium: an antisigma factor inhibits the activity of    the flagellum-specific sigma factor, sigma F. Mol. Microbiol.    6:3149-3157.-   42. Osterberg, S., T. del Peso-Santos, and V. Shingler. 2011.    Regulation of alternative sigma factor use. Annu. Rev. Microbiol.    65:37-55.-   43. Paul, K., M. Erhardt, T. Hirano, D. F. Blair, and K. T.    Hughes. 2008. Energy source of flagellar type III secretion. Nature    451:489-492.-   44. Singer, H. M., M. Erhardt, A. M. Steiner, M. M. Zhang, D.    Yoshikami, G. Bulaj, B. M. Olivera, and K. T. Hughes. 2012.    Selective purification of recombinant neuroactive peptides using the    flagellar type III secretion system. MBio 3.-   45. Sorenson, M. K., S. S. Ray, and S. A. Darst. 2004. Crystal    structure of the flagellar sigma/anti-sigma complex sigma(28)/FlgM    reveals an intact sigma factor in an inactive conformation. Mol.    Cell 14:127-138.-   46. Sourjik, V., and N. S. Wingreen. 2012. Responding to chemical    gradients: bacterial chemotaxis. Curr. Opin. Cell Biol. 24:262-268.-   47. Takaya, A., M. Erhardt, K. Karata, K. Winterberg, T. Yamamoto,    and K. T. Hughes. 2012. YdiV: a dual function protein that targets    FlhDC for ClpXP-dependent degradation by promoting release of    DNA-bound FlhDC complex. Mol. Microbiol. 83:1268-1284.-   48. Tomoyasu, T., T. Ohkishi, Y. Ukyo, A. Tokumitsu, A. Takaya, M.    Suzuki, K. Sekiya, H. Matsui, K. Kutsukake, and T. Yamamoto. 2002.    The ClpXP ATP-dependent protease regulates flagellum synthesis in    Salmonella enterica serovar Typhimurium. J. Bacteriol. 184:645-653.-   49. Wada, T., T. Morizane, T. Abo, A. Tominaga, K. Inoue-Tanaka,    and K. Kutsukake. 2011. EAL domain protein YdiV acts as an    anti-FlhD4C2 factor responsible for nutritional control of the    flagellar regulon in Salmonella enterica Serovar Typhimurium. J.    Bacteriol. 193:1600-1611.-   50. Wang, Q., Y. Zhao, M. McClelland, and R. M. Harshey. 2007. The    RcsCDB signaling system and swarming motility in Salmonella enterica    serovar Typhimurium: dual regulation of flagellar and SPI-2    virulence genes. J. Bacteriol. 189:8447-8457.-   51. Wang, S., R. T. Fleming, E. M. Westbrook, P. Matsumura,    and D. B. McKay. 2006. Structure of the Escherichia coli FlhDC    complex, a prokaryotic heteromeric regulator of transcription. J.    Mol. Biol. 355:798-808.-   52. Wei, B. L., A. M. Brun-Zinkernagel, J. W. Simecka, B. M.    Pruss, P. Babitzke, and T. Romeo. 2001. Positive regulation of    motility and flhDC expression by the RNA-binding protein CsrA of    Escherichia coli. Molecular microbiology Mol. Microbiol. 40:245-256.-   53. Wozniak, C. E., C. Lee, and K. T. Hughes. 2009. T-POP array    identifies EcnR and PefI-SrgD as novel regulators of flagellar gene    expression. J. Bacteriol. 191:1498-1508.-   54. Yamamoto, S., and K. Kutsukake. 2006. FliT acts as an    anti-FlhD2C2 factor in the transcriptional control of the flagellar    regulon in Salmonella enterica serovar Typhimurium. J. Bacteriol.    188:6703-6708.-   55. Yanagihara, S., S. Iyoda, K. Ohnishi, T. Iino, and K.    Kutsukake. 1999. Structure and transcriptional control of the    flagellar master operon of Salmonella typhimurium. Genes Genet.    Syst. 74:105-111.-   56. Yokoseki, T., K. Kutsukake, K. Ohnishi, and T. Iino. 1995.    Functional analysis of the flagellar genes in the fliD operon of    Salmonella typhimurium. Microbiol. 141:1715-1722.

1-49. (canceled)
 50. A recombinant cell line comprising one or moremutations in a gene selected from the group consisting of fliY, fliF,flgM, araBAD, fliA, fliC, fliD, prgH-hilA, cheV, tcp, yhjH, aer, mcpC,PmotA, motA-cheZ, flhDC, flgM, flgN, flgKL, trg, ycgR, mcpA, fliB, tsr,ecnR, hin-fljA, mcpB, lrhA, ydiV, flgE, and fljB; wherein therecombinant cell line is a Salmonella enterica or an Escherichia colicell line.
 51. The recombinant cell line of claim 50, wherein the cellline is a strain selected from the group consisting of TH2788, TH4885,TH1539, TH10874, TH15360, TH15705, TH15706, TH15707, TH16229, TH16240,TH16778, TH17020, EM170, EM171, EM172, EM173, EM174, EM175, EM176,EM177, EM178, and EM179.
 52. The recombinant cell line of claim 50,further comprising a vector comprising a FlgM nucleic acid sequenceoperably linked to a nucleic acid sequence encoding a purification tag,a cleavage site, a nucleic acid sequence of interest, and atranscription control element (TCE); wherein the TCE is heterologous tothe FlgM nucleic acid sequence; and wherein the 5′ to 3′ order of thesequences is the FlgM nucleic acid sequence, the nucleic acid sequenceencoding a purification tag, the cleavage site, and the nucleic acidsequence of interest.
 53. The recombinant cell line of claim 50, whereinthe one or more mutations is in a coding region of the gene.
 54. Therecombinant cell line of claim 50, wherein the one or more mutations isin a ribosome binding sequence (RBS) of the gene.
 55. The recombinantcell line of claim 50, wherein the one or more mutations is in apromoter of the gene.
 56. A recombinant cell line of claim 50, whereinthe one or more mutations is a mutation in fliA.
 57. The recombinantcell line of claim 56, wherein the one or more fliA mutations isselected from the group consisting of V33E; L199R H14N; H14D; T138I;E203D; R91C; L207P; and a fliA start codon mutation from GTG to ATG. 58.The recombinant cell line of claim 57, wherein the one or more fliAmutation is selected from the group consisting of: (a) a double mutantof R91C and L207P; (b) a triple mutant of H14D, R91C, and L207P; (c) adouble mutant of fliA start codon change from GTG to ATG and H14N; and(d) a double mutant of fliA start codon change from GTG to ATG and amutation in the fliA ribosome binding sequence (RBS) to the canonicalsequence (CRBS).
 59. The recombinant cell line of claim 50, wherein thebackground strain is derived from a fljBenx Vh2 mutant strain.
 60. Therecombinant cell line of claim 52, wherein the purification tagcomprises poly-histidine, glutathione S-transferase (GST), Myc, HA,FLAG, or maltose binding protein (MBP).
 61. The recombinant cell line ofclaim 52, wherein the FlgM nucleic acid sequence is wild type FlgM. 62.The recombinant cell line of claim 52, wherein the cleavage sitecomprises a Tobacco Etch Virus (TEV) protease cleavage site or anEnterokinase (ETK) cleavage site.
 63. The recombinant cell line of claim52, wherein the nucleic acid sequence of interest encodes acysteine-rich peptide.
 64. The recombinant cell line of claim 63,wherein the cysteine-rich peptide comprises a neuroactive toxin.
 65. Therecombinant cell line of claim 52, wherein the TCE is a constitutive TCEor a regulatable TCE.
 66. The recombinant cell line of claim 65, whereinthe regulatable TCE comprises an inducible promoter.
 67. The recombinantcell line claim 66, wherein the inducible promoter comprises aP_(araBAD) promoter.
 68. A FlgM peptide produced by the recombinant cellline of claim
 52. 69. The FlgM peptide of claim 68, wherein thepurification tag comprises poly-histidine.
 70. A method of producing apeptide of interest comprising culturing the recombinant cell line ofclaim 52 in culture media.
 71. The method of claim 70, wherein thebacterial host cell is cultured in media comprising about 200 mM toabout 400 mM NaCl or KCl and wherein more of the peptide of interest isproduced as compared to bacterial host cells cultured in mediacomprising 100 mM NaCl or KCl, respectively.